PSC Resource Handbook – Reprogramming
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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 associated with varying levels of efficiency and safety (Figure 1.1).
To find the best solution for your reprogramming experiment, go to thermofisher.com/reprogramming
Figure 1.1. Safety and efficiency of various reprogramming technologies. Different reprogramming agents are classified as integrating, excisable, or nonintegrating technologies, which are associated with increasing levels of safety. Under each category, technologies are listed in order of decreasing efficiency
Traditional reprogramming technologies using lentiviruses or retroviruses involve 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 nonintegrating technologies because they avoid the issue of insertional mutations and generate footprint-free PSCs that do not contain detectable vectors or transgenes.
Two common nonintegrating reprogramming technologies make use of episomal vectors and Sendai virus (SeV). These two technologies are discussed in more detail in this section. Other nonintegrating reprogramming technologies make use of mRNAs, miRNAs, proteins, and other small molecules.
1.2 Choosing a reprogramming method
Different reprogramming technologies have their own advantages and disadvantages that must be weighed when planning an experiment. The main issues 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. Features of the Invitrogen Epi5 iPSC Episomal Reprogramming Kit and Gibco CytoTune-iPS 2.0 Sendai Reprogramming Kit are compared in Table 1.1. Generally, the CytoTune reprogramming kit is 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 1.1. Episomal and Sendai reprogramming features and selection guide.
- Parental fibroblasts used for reprogramming should be from an early passage (<P6) with normal growth and karyotype.
- The density of seeded fibroblasts prior to initiation of reprogramming is critical to achieving good reprogramming efficiencies. Confluence of 50–80% is recommended on the day of transfection or transduction.
- Protocols describe reprogramming in 6-well formats. The protocol can be scaled down to a 12-well or 24-well culture dish, albeit with potentially reduced efficiency.
- Besides fibroblasts, a variety of somatic cells can be used for reprogramming. The CytoTune-iPS 2.0 Sendai Reprogramming Kit has been validated for a wide variety of cell types, including:
- Adult and neonatal dermal fibroblasts
- Amniotic fluid MSCs
- Cardiac fibroblasts
- CD34+ blood cells
- Mammary epithelial cells
- Mouse embryonic fibroblasts
- Nasal epithelial cells
- Peripheral blood mononuclear cells (PBMCs)
- Skeletal myoblasts
- T cells
- Umbilical vein epithelial cells
- Urine epithelial cells
- The Epi5 and CytoTune reprogramming systems are validated for human cells. oriP/EBNA1 vectors are also known to function in canine cells, while their use in murine systems may require additional components. For a current list of publications citing the use of SeV for reprogramming various cell types and species, go to thermofisher.com/sendaipubs
- 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 with Gibco KnockOut Serum Replacement–based media for feeder-dependent systems and Gibco Essential 8 Medium for feeder-free reprogramming at thermofisher.com/reprogramprotocols
- 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, we recommend testing different seeding densities using at least two or three different multiplicity of infection values (e.g., 1, 3, and 9) of the Invitrogen CytoTune EmGFP Sendai Fluorescence Reporter.
- The expression of EmGFP in successfully transduced cells is detectable at 24 hours posttransduction by fluorescence microscopy, and reaches maximal levels at 48–72 hours posttransduction. Note that cells infected with SeV will most likely be refractive to further infection. Therefore, trying to use the CytoTune-iPS 2.0 Sendai Reprogramming Kit with cells already transduced with the CytoTune EmGFP Sendai Fluorescence Reporter, or vice versa, are not recommended.
1.3 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 transfection . 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 manipulation . 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 efficiencies [3,4], with the mp53DD dominant negative mutant providing higher-efficiency knockdown compared to traditional short hairpin RNA (shRNA) systems . An improved reprogramming system described by Okita et al.  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 1.2). 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.
Figure 1.2. 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 (Figure 1.3).
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.
Figure 1.3. A comparison of Sendai and episomal vector reprogramming workflows, starting from various somatic cell types.
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 cell [7-9]. 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/TS15ΔF), enables safe and effective delivery and expression of reprogramming genes [7-10]. 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. . The CytoTune-iPS 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 containing additional Klf4 to achieve higher reprogramming efficiency (Figure 1.4). The CTS CytoTune-iPS 2.1 Sendai Reprogramming Kit also contains three vectors, KOS and Klf4 as in the 2.0 kit, and the third carrying L-Myc, which is reported to be less oncogenic than c-Myc (Table 1.2). The Sendai vectors offered in the CytoTune kits are efficient at reprogramming fibroblasts, PBMCs, CD34+ cells, and T cells (workflows shown in Figure 1.3).
Figure 1.4. Configuration of vectors in the CytoTune 2.0 Reprogramming Kit.
Table 1.2 Comparison of reprogramming kits.
While many researchers choose to begin reprogramming with dermal fibroblasts (Figure 1.5), the CytoTune kit has proven to be effective at reprogramming multiple somatic cell types, including PBMCs, CD34+ cells, and T cells (Figure 1.6).
Figure 1.5. A time course of iPSC colony formation. Cells were replated at day 7. Initial colony formation is usually visible by days 10–12. By day 21, large compact colonies will emerge from the background lawn of fibroblasts.
Figure 1.6. iPSCs were derived from various somatic cell sources using the CytoTune 2.0 kit and were grown under feeder-free conditions using Essential 8 Medium in wells coated with Gibco Geltrex matrix or vitronectin (VTN) or rhLaminin-521 (LN521). The resulting colonies were stained for alkaline phosphatase expression at day 19–23.
Identification of colony quality
Two primary methods of distinguishing a “good” colony from a “bad” colony include morphology and live staining (Figure 1.7). As clones are established, residual Sendai virus is cleared from the population. In general, Sendai vector clearance is complete by passage 10 (Figure 1.8).
After a stable iPSC line has been generated, it is critical to demonstrate the quality and utility of the line before proceeding to differentiation. Assays such as immunofluorescence, qPCR, and flow cytometry are useful readouts for the ability of the line to produce cells of all three embryonic germ layers (Figures 1.9 and 1.10). Traditional cytogenetic analysis is important to demonstrate normal karyotype (Figure 1.9).
Figure 1.9. Two representative iPSC clones, derived using the CytoTune 2.0 kit at MOI of 5-5-3 (top panel) or 10-10-3 (bottom panel), show pluripotent marker expression (Nanog), normal G-banding karyotype, and the ability to differentiate into representative germ layers as assessed using an Applied Biosystems TaqMan hPSC Scorecard Panel.
Figure 1.10. A representative CytoTune 2.0 derived iPSC clone was subjected to directed differentiation into (A) neural stem cells using Gibco PSC Neural Induction Medium, (B) definitive endoderm using the Gibco PSC Definitive Endoderm Differentiation Kit, or (C) cardiomyocytes using the Gibco PSC Cardiomyocyte Differentiation Kit.
- 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.
- 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.
- 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.
- Spike BT, Wahl GM (2011). p53, stem cells, and reprogramming: tumor suppression beyond guarding the genome. Genes Cancer 2(4):404–419.
- Kawamura T, Suzuki J, Wang YV, et al (2009). Linking the p53 tumour suppressor pathway to somatic cell reprogramming. Nature 460(7259):1140–1144.
- 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.
- 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.
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- 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.
- 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.
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