Transposon‐Based Cellular Reprogramming to Induced Pluripotency

Abstract

Induced pluripotent stem (iPS) cells are a seminal discovery in the field of stem cells. iPS cells can be employed as patient‐specific pluripotent stem cells in disease modelling, drug screens and potentially for autologous cell therapy, without the ethical concerns associated with human embryonic stem (ES) cells. Initially, iPS cells were generated by viral transduction of fibroblasts with core reprogramming genes, such as Oct4, Sox2, Klf4 and c‐Myc. However, integrating viruses may cause insertional mutagenesis and may increase the risk of tumour formation. Therefore, alternative gene transfer approaches which avoid these safety concerns are required. DNA (deoxyribonucleic acid) transposons are nonviral genetic elements, which can be designed as efficient vectors for gene transfer. DNA transposons are distinguished by their large cargo capacity, their relatively simple design and the availability of transposase variants. Transposon‐based reprogramming broadens the toolbox for the generation of iPS cell lines and will advance the development of safe, nonviral methods.

Key Concepts

  • During mammalian ontogenesis, cellular potency is gradually lost.
  • Mammalian ontogenesis is a one‐way process from pluripotent to somatic cells.
  • Experimentally, somatic cells can be reprogrammed to a state of induced pluripotency by the transduction of core reprogramming factors.
  • DNA transposons are a promising nonviral approach for reprogramming.
  • DNA transposon‐derived iPS cells can be triggered to differentiate in vitro and in vivo.

Keywords: transposition; Sleeping Beauty; piggyBac; stemness; ontogenesis; synthetic biology; large animal models

Figure 1. Mammalian ontogenesis and cellular reprogramming. (a) During ontogenesis, pluripotent stem cells progress to progenitor cells, which develop to terminally differentiated cells. During this one‐way process, the cells lose their potency. (b) Experimentally, differentiated, somatic cells can be reprogrammed, for example, by the simultaneous viral transduction of the core reprogramming genes Oct4, Sox2, Klf4 and c‐Myc.
Figure 2. DNA transposons and nonautonomous transposon systems. (a) Naturally occurring DNA (deoxyribonucleic acid) transposons (jumping genes) consist of a transposase gene flanked by inverted terminal repeats (ITRs). Upon expression of the transposase, the protein binds to the ITRs and cuts the whole element out of the original site. In a second step, the transposon DNA becomes integrated in another genomic site (cut‐and‐paste mechanism). As long as the transposase protein is present, the transposon will hop around. (b) In engineered, nonautonomous DNA systems, the ITRs and the transposase gene are separated on two bacterial plasmids. Any DNA, for example a reprogramming factor, can now be inserted between the ITRs. After coelectroporation or colipofection of both plasmids into a mammalian cell, the transposase will become expressed and subsequently transpose the ITR‐flanked cargo DNA into the genome. The episomal transposase plasmid will be degraded, and the transposon is then fixed in a certain site of the genome.
Figure 3. Transposon‐based reprogramming. The transposon plasmid carries an ITR‐flanked reprogramming construct of a strong promoter driving a multigene arrangement of reprogramming factors. The transposase (helper) plasmid carries the corresponding transposase gene. Upon cotransduction into mammalian cells, the transposase gene is expressed and catalyses integration of the ITR‐flanked transposon into the genome, resulting in robust expression of the reprogramming genes. After reprogramming to a pluripotent state, the transposon cassette can be removed by transduction of an excision‐competent, integration‐deficient transposase variant (Yusa et al., ). The pluripotent state is then maintained by endogenous stemness factors.
Figure 4. Potential autologous cell therapy based on iPS cells. Somatic cells are obtained from a patient and are reprogrammed to iPS cells in vitro. Owing to the unique, unlimited self‐renewal capacity and pluripotent state of the iPS cells, the required cell numbers can be amplified and then triggered to differentiate into desired cell types, for example hepatocytes, haematopoietic cells or neurons. Potentially, the derived differentiated cells can be transplanted in the patient to restore specific functions. In an autologous setting, no immune‐suppressive treatment is required.
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Kues, Wilfried A(Nov 2016) Transposon‐Based Cellular Reprogramming to Induced Pluripotency. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0026889]