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3.1 Introduction

iPSCs are generated from somatic cells through the forced expression of specific transcription factors that reprogram the cells to a pluripotent state. To date, different sets of reprogramming factors have been tested, along with different types of gene delivery technologies that are associated with varying levels of efficiency and safety (Figure 3.1).

Safety efficiency of various reprogramming technologies
Figure 3.1. Safety and efficiency of various reprogramming technologies. Different reprogramming agents are classified as integrating, excisable or non-integrating technologies, which exhibit increasing levels of safety. Under each category, technologies are listed in order of decreasing efficiency.

 

Traditional reprogramming technologies using lentivirus or retrovirus involve the integration of foreign DNA into the host genome. This can lead to insertional mutagenesis, which can affect the properties of the derived cell lines. The general trend in the field has been towards non-integrating technologies because they avoid the issue of insertional mutations and generate footprint-free PSCs that do not contain detectable vectors or transgenes.

Two common non-integrating reprogramming technologies make use of episomal vectors and Sendai virus (SeV). These two technologies are discussed in more detail in this section Other non-integrating reprogramming technologies make use of mRNAs, miRNAs, proteins and other small molecules.

Find the best solution for your reprogramming experiment


3.2 Choosing a reprogramming method

Different reprogramming technologies have their own advantages and disadvantages that must be weighed when planning an experiment. The main features to consider include a lab’s flexibility in working with viruses, the intended parental somatic cells, the efficiency required in downstream experiments, and the importance of avoiding any chance of genomic integration. These features are compared between the Invitrogen Epi5 Reprogramming Kit  and Gibco CytoTune-iPS Sendai and CytoTune-iPS 2.0 Sendai reprogramming  kits in Table 3.1. Generally, CytoTune reprogramming kits are great for parental cells that are difficult to reprogram and for experiments that require higher efficiency reprogramming and footprint-free iPSCs Epi5 Reprogramming Vectors  work well for parental cells that are easy to reprogram, especially when viral particles cannot be used.

Table 3.1. Episomal and Sendai reprogramming features and selection guide.

 

Epi5 iPSC Reprogramming Kit

CytoTune and CytoTune 2.0 -iPS Sendai Reprogramming Kits

Description Virus-free non-integrating episomal DNA vectors Non-integrating RNA virus
Reprogramming efficiency 0.01–0.1% 0.05–1%
Genomic integration-free Yes, but all DNA vectors have a minor chance of integration Yes
Virus-free reprogramming Yes No
Blood cell reprogramming Yes, for limited cell types (CD34+ cells) and with low efficiency Yes, for many cell types (CD34+ cells, PBMCs, T cells) and with high efficiency
Special equipment required Neon Transfection System or similar device for blood reprogramming; Lipofectamine 3000 can be used with fibroblasts None
Reprogramming factors Oct4, Sox2, Nanog, Lin28, Klf4, and L-Myc Oct4, Sox2, Klf4, c-Myc
Kit format

2 tubes with 20 μl each:

Tube A: mixture of pCE-hOCT3/4, pCE-hSK (containing Sox2, Klf4), and pCE-hUL (containing L-Myc, Lin28)

Tube B: mixture of pCE-mP53DD and pCXB-EBNA1

CytoTune Kit

4 tubes with 100 μl each:

  • CytoTune Sendai hOct3/4
  • CytoTune Sendai hSox2
  • CytoTune Sendai hKlf4
  • CytoTune Sendai hc-Myc
     

CytoTune 2.0 Kit

3 tubes with 100 μl each:

  • CytoTune 2.0 KOS (containing Klf4, Oct3/4, and Sox2)
  • CytoTune 2.0 hc-Myc
  • CytoTune 2.0 hKlf4
Transfection/ transduction control
None
CytoTune EmGFP Sendai Fluorescence Reporter
Detection of residual reprogramming vector backbones Endpoint PCR qPCR, endpoint PCR, or TaqMan® hPSC Scorecard Panel

Useful tips

  • Parental fibroblasts used for reprogramming should be early passage (<P6) with normal growth and karyotype
  • Density of seeded fibroblasts prior to initiation of reprogramming is critical to achieve good reprogramming efficiencies. A confluence of 50–80% is recommended on the day of transfection or transduction
  • Protocols describe reprogramming in 6-well formats Protocol can be scaled down to a 12-well or 24-well culture dish, albeit with a potentially reduced efficiency
  • Besides fibroblasts, a variety of somatic cells can be used for reprogramming Gibco CytoTune-iPS 2.0 Sendai Reprogramming Kit  has been validated for a wide variety of cell types, including human fibroblasts, CD34+ cord blood cells, and peripheral blood mononuclear cells. 
    Find protocols for reprogramming these cell types
  • The Epi5 and CytoTune reprogramming systems are validated for human cells. EBNA/oriP vectors are also known to function in canine cells, while their use in murine systems may require additional components.
    See publications citing the use of SeV for reprogramming
  • Reprogramming can be carried out on either feeder-dependent or feeder-free culture systems. Typically the efficiency of reprogramming is higher in feeder-dependent systems than under feeder-free conditions because of the more nutrient-rich formulations. 
    View validated protocols for Epi5 and CytoTune reprogramming
  • CytoTune 2 0 reprogramming can be optimized for maximal reprogramming efficiency by varying the amount of Klf4. Typically, the multiplicity of infection for the KOS and c-Myc vectors are maintained at a 1:1 ratio, with the Klf4 vector varied independently. The standard ratio for KOS:c-Myc:Klf4 is 5:5:3, which could be changed to 5:5:6 or 10:10:6 to achieve higher efficiency
  • To optimize transduction of hard-to-transduce cells, it is recommended to test different seeding densities using at least two or three different multiplicity of infection values (e g , 1, 3, and 9) of Invitrogen CytoTune EmGFP Sendai Fluorescence Reporter. The expression of EmGFP in successfully transduced cells is detectable at 24 hours post-transduction by fluorescence microscopy, and reaches maximal levels at 48–72 hours post-transduction. Note that cells infected with SeV will most likely be refractive to further infection. Therefore, it is not recommended to try and use the CytoTune-iPS 2 0 Sendai Reprogramming Kit with cells already transduced with the CytoTune EmGFP Sendai Fluorescence Reporter or vice versa

3.3 Reprogramming with episomal vectors

Episomal vectors are circular extrachromosomal DNA molecules that are used to introduce and express exogenous genetic material. They are attractive reprogramming vectors because they carry viral elements that allow the prolonged and controlled expression of reprogramming factors, but they can be transfected into cells without the need for viral packaging.

One popular episomal vector system specifically incorporates the oriP/EBNA1 system derived from the Epstein-Barr virus. The oriP sequence is a cis-acting element that serves as the origin of replication on the pCEP backbone of the reprogramming vectors; EBNA1 codes for a DNA-binding protein that binds to oriP and tethers the plasmids to genomic DNA during replication, allowing one replication per cycle. Together, the oriP and EBNA1 elements ensure the replication and retention of the reprogramming vectors during each cell division, driving high expression of reprogramming genes and allowing iPSC derivation in a single transfection1. The loss of the episomal vectors at a rate of ~5% per cell cycle allows the removal of vectors from the iPSCs without any additional manipulation2. Therefore, while reprogramming vectors are retained long enough for reprogramming to occur, they are lost over time, so the newly derived iPSCs are footprint-free, lacking transfected DNA and integrated transgenes.

Knockdown of p53 has been shown to improve reprogramming efficiencies3, 4, with the mp53DD dominant negative mutant providing higher efficiency knockdown compared to traditional shRNA systems5. An improved reprogramming system described by Okita et al 6 includes episomal vectors carrying reprogramming factors along with mp53DD. In this system, an additional EBNA1 expression vector ensures high expression of reprogramming factors at the early stages of reprogramming.

A complete set of vectors based on the above study is available in the Epi5 Episomal iPSC Reprogramming Kit (Figure 3.2). The kit includes two tubes: a reprogramming vector tube containing a mixture of three plasmids that code for Oct3/4, Sox2, Klf4, L-Myc and Lin28; and a second tube containing a mixture of two plasmids that code for the p53 dominant negative mutant and EBNA1.

Configuration of vectors Epi5 Reprogramming Kit
Figure 3.2. Configuration of vectors in the Epi5 Reprogramming Kit

 

With all of these vectors together, the Epi5 Reprogramming System achieves efficiencies of around 0.01 to 0.1% and can be used to reprogram different cell types, including CD34+ blood cells. To initiate reprogramming, the kit must be used in combination with a gene delivery system.

The Invitrogen Neon Transfection System allows electroporation of the vectors into most cell types. For fibroblasts, it is possible to achieve efficient reprogramming without electroporation through the use of Invitrogen Lipofectamine 3000 Transfection Reagent.


3.4 Reprogramming with Sendai virus (SeV)

SeV is an enveloped virus with a single-chain RNA genome in the minus sense. This genome codes for the structural proteins that form and support the envelope (NP and M); the subunits of RNA polymerase (P and L); hemagglutinin-neuraminidase (HN), which recognizes sialic acid; and fusion protein (F), which, when activated by a protease, fuses the viral envelope with the cell membrane during infection.

There are two main characteristics that make SeV an attractive system for reprogramming. First, it can infect a wide range of cell types from various animal species because SeV infects cells by attaching itself to the sialic acid present on the surface of many different cells. Second, the SeV vectors are made of RNA and remain in the cytoplasm, ensuring that they do not integrate into the host genome or alter the genetic information of the host cell7-9. This is in contrast to retroviral vectors that require integration into host chromosomes to express reprogramming genes or even adenovirus and plasmid vectors that exist episomally and do not require integration but carry the possibility of integrating into host chromosomes by virtue of being DNA-based SeV, modified through deletion of the F gene and introduction of temperature sensitivity mutations in SeV proteins (SeV/TSΔF and SeV/TS15 ΔF), enables safe and effective delivery and expression of reprogramming genes7-10 . These modifications prevent transmission and curtail the propagation of the reprogramming vectors. Thus, the viral vectors contained in the cytoplasm are eventually diluted out, leaving footprint-free iPSCs. 

Currently, there are two CytoTune reprogramming kits based on the SeV system developed by Fusaki et al 7. The CytoTune-iPS Reprogramming Kit contains four SeV-based reprogramming vectors, each capable of expressing one of the four Yamanaka factors (i e, Oct4, Sox2, Klf4, and c-Myc) (Figure 3.3).  The more recent CytoTuneiPS 2.0 Sendai Reprogramming Kit contains only three vectors, the first one combining Oct4, Sox2,and Klf4; the second one containing c-Myc; and the third one contributing additional Klf4 and RNA polymerase to achieve higher reprogramming efficiency (Figure 3.3). The key differences between the two kits are highlighted in Table 3.2.

Configuration of vectors in the CytoTune Reprogramming Kits.

Figure 3.3. Configuration of vectors in the CytoTune Reprogramming Kits.


Table 3.2 Comparison of reprogramming kits.

  CytoTune kit CytoTune 2.0 kit
Efficiency + +++
Cytotoxicity ++ +
Viral clearance (safety) ~P10 ~P3

 

References

  1. Yu J, Hu K, Smuga-Otto K, et al (2009). Human induced pluripotent stem cells free of vector and transgene sequences. Science 324(5928):797–801
  2. Nanbo A, Sugden A, Sugden B (2007). The coupling of synthesis and partitioning of EBV’s plasmid replicon is revealed in live cells. EMBO J 26(19):4252–4262
  3. Hong H, Takahashi K, Ichisaka T, et al (2009). Suppression of induced pluripotent stem cell generation by the p53-p21 pathway. Nature 460(7259):1132–1135
  4. Spike BT, Wahl GM (2011). p53, stem cells, and reprogramming: tumor suppression beyond guarding the genome. Genes Cancer 2(4):404–419.
  5. Kawamura T, Suzuki J, Wang YV, et al (2009). Linking the p53 tumour suppressor pathway to somatic cell reprogramming. Nature 460(7259):1140–1144.
  6. Okita K, Yamakawa T, Matsumura Y, et al (2013). An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells. Stem Cells 31(3):458–466
  7. Fusaki N, Ban H, Nishiyama A, et al (2009). Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc Jpn Acad Ser B Phys Biol Sci 85(8):348–362.
  8. Li HO, Zhu YF, Asakawa M, et al (2000). A cytoplasmic RNA vector derived from non-transmissible Sendai virus with efficient gene transfer and expression. J Virol 74(14):6564–6569.
  9. Seki T, Yuasa S, Oda M, et al (2010). Generation of induced pluripotent stem cells from human terminally differentiated circulating T cells. Stem Cell 7(1):11–14.
  10. Inoue M, Tokusumi Y, Ban H, et al (2003). Non-transmissible virus-like particle formation by F-deficient Sendai virus is temperature sensitive and reduced by mutations in M and HN proteins. J Virol 77(5):3238–3246.