Introduction

Advances in cell reprogramming technology has catapulted the field of stem cell biology and its applications in disease research and therapeutic development, to where it is today. The initial discovery that adult somatic cells could be reprogrammed into human induced pluripotent stem cells (hiPSCs) (Takahashi et al. 2007; Scudellari 2016) has made pluripotent stem cell biology a flourishing research area. Cell reprogramming is not only enabling a better understanding of human disease pathways, but is improving the reliability of in vitro drug screening to boost the translatability of disease research into therapies that can directly help patients.

For example, cell reprogramming has led to the creation of hiPSCs carrying the genes of patients with specific disease types, offering researchers an innovative model to discover personalized treatments and further advancing the field of personalized medicine. Moreover, in disease therapy, autologous hiPSC transplants are being used to replace diseased or ineffective cells with reprogrammed healthy new cells, offering lifesaving opportunities to patients with serious blood disorders.

However, the clinical applications of this technology have only been possible since the crucial discovery of non-integrating reprogramming technologies. Unlike traditional integrating methods, these non-integrating systems are able to generate ‘clean’ hiPSCs that contain no trace of the vectors used to reprogram the cells, and so do not result in mutations within the host cell genome (Kang et al. 2015). As such, non-integrating reprogramming of human somatic cells has now become the gold standard approach for clinical applications.

Below we review the different non-integrating reprogramming systems available to researchers along with the approaches we use at Axol to generate hiPSCs from your cells. This custom service gives you the freedom to choose from different reprogramming technologies, cell origins, and culture systems, so you can acquire a rapid, reliable and cost-effective supply of relevant hiPSCs that are best suited to your research objectives.

What are the benefits and limitations of different non-integrating reprogramming technologies?

The three most widely used non-integrating reprogramming systems, episomal vector (Epi), Sendai-virus (SeV) and mRNA transfection technologies, all generate high-quality hiPSCs, but differ in a number of other characteristics (Schlaeger et al. 2015). For example, although mRNA reprogramming appears to be the most efficient (in terms of the number of emerging hiPSC colonies per starting cell number), the SeV and Epi methods showed significantly higher success rates (the number of experiments yielding at least three hiPSC colonies) than the mRNA method (Schlaeger et al. 2015).

The same study also found that SeV and Epi methods required significantly less hands-on time to produce ready-to-use colonies compared to the mRNA method (SeV=3.5 hours, Epi=4 hours and mRNA=8 hours). Additionally, the researchers conducted a survey of independent human reprogramming laboratories, and found that a high proportion (47%) of the most experienced laboratories reported that they had been unable to generate RNA-hiPSCs, suggesting that the difficulties with using the RNA method are not dependent on the laboratory’s expertise (Schlaeger et al. 2015).

Which reprogramming technologies should you use?

For the reasons stated above, at Axol, we use the Epi and SeV technologies to ensure we supply you with a fast, reliable and translatable source of hiPSCs. However, before you decide which one to use, it is essential to consider which of these technologies is best suited to your specific research needs.

Episomal vector reprogramming

Epi technology uses Epstein-Barr virus-derived sequences to facilitate episomal plasmid DNA replication in dividing cells (Yu et al. 2007). The Epi method has a high success rate in generating hiPSCs from healthy or diseased patient donor peripheral blood mononuclear cells (PBMCs) and fibroblast cells, with its reprogramming efficiency having undergone extensive improvements since its conception (Okita et al. 2011; Chou et al. 2011).

The resulting Epi-hiPSCs are ‘footprint-free’ because they exhibit a quick loss of reprogramming vectors, particularly when reprogramming fibroblasts (Schlaeger et al. 2015). This therefore offers an extremely reliable and efficient reprogramming strategy.

This non-viral reprogramming method utilizes Professor Yamanaka’s 4-factor reprogramming capabilities (Takahashi et al. 2007) and is licensed to Axol from iPS Academia, Japan. It does not require a Category II Tissue Culture Laboratory nor a license for derivatives of the iPSCs.

Sendai-virus reprogramming

Additionally, we can produce hiPSCs using the CytoTune®-iPS 2.0 Sendai-virus (SeV) reprogramming technology (ID Pharma, Japan). Uniquely (as an Axol customer), if you do not have an ID Pharma SeV technology license, you can obtain a project-specific sub-license as part of our cell reprogramming contract service. Additionally, given that the SeV method is licensed under clear regulatory and intellectual property (IP) guidelines, large pharmaceutical companies find this method particularly appealing.

Although it is a viral method, the Sendai-virus technology is non-integrating, because the RNA virus does not enter the nucleus of the donor cell, so is typically diluted out of cells after 10 passages post-infection (Malik & Rao 2013) (however, this does mean that the SeV method is a lot slower than the Epi method at generating hiPSCs).

The SeV method has been found to be reliable and more efficient than Epi (Schlaeger et al. 2015), requiring fewer starting cells and reprogramming cells within approximately 25 days at an efficiency of 0.1% for PBMCs and 1% for fibroblasts (Malik & Rao 2013). It also requires less time to generate ready-to-use colonies, and SeV-hiPSCs exhibit significantly lower aneuploidy rates than Epi-hiPSCs (4.6% versus 11.5%) (Schlaeger et al. 2015).

Feeder-layer or feeder-free culture system?

You have the choice of using either a feeder-free or feeder-layer culture system to generate hiPSCs. The feeder-layer system uses a layer of inactive murine embryonic fibroblast (MEF) feeder cells during reprogramming, whereas the feeder-free system uses defined reagents from non-animal sources. This is coupled with extracellular matrices to support cell survival, adhesion, and pluripotency, along with factors that promote proliferation. Both systems offer different benefits, so it’s important to consider which culture system best suits your research requirements (Table 1).

Table 1 : The benefits of using feeder-layer and feeder-free systems for culturing reprogrammed cells using the Sendai-virus and Episomal vector reprogramming methods.
Feeder-layer system Feeder-free system
Increases the number of colonies produced Eliminates the need to use animal materials
Once generated, colonies can be sustained as a stable pluripotent culture for a prolonged period of time in vitro Better reliability in the culture medium and reprogramming processing, due to the removal of variable components in animal materials
Higher quality of hiPSCs Cost-effective – no requirement to purchase feeder cells
Once generated, the hiPSCs can be adapted to a feeder-free system for easy maintenance, or to a feeder-layer system Easier maintenance
Reduces culture time, because there is no need for cells to adapt to a feeder layer

Which cell source should you use?

Many sources of somatic cells are used for generating hiPSCs, such as peripheral blood mononuclear cells (PBMCs), neuronal progenitor cells, keratinocytes, hepatocytes, but these cell sources vary in ease, efficacy, and cost (El Hokayem et al. 2016). We routinely offer reprogramming of two different cell types that are the most easily accessible and reliable in terms of success rates: fibroblasts and PBMCs.

Episomal vector reprogramming

Dermal fibroblasts were the first cell type to be successfully reprogrammed into hiPSCs (Takahashi et al. 2007) and fibroblasts are still widely used and accepted as the gold standard for reprogramming efficiency and differentiation (Li et al. 2014).

Alternatively, PBMCs are emerging as an effective and easily accessible, non-invasive source for patient-specific hiPSC derivation (El Hokayem et al. 2016). A variety of studies have demonstrated the feasibility of using PBMCs as an accessible resource for cell reprogramming and modeling some diseases, such as Alzheimer’s disease (Yu et al. 2007; Táncos et al. 2016).

There are comparative advantages and disadvantages of using fibroblasts versus PBMCs as the cell source for reprogramming (Table 2) (El Hokayem et al. 2016). We recommend weighing these up before deciding which one to use for your specific research project.

Sendai-virus reprogramming

Whichever cell type you choose to reprogram, you can use our custom cell sourcing service or ready supply of cryopreserved fibroblasts or blood cells . Additionally, both types of cells can be used with either the Epi or SeV reprogramming technologies.

Table 2 : The advantages (+) and disadvantages (-) of using fibroblasts and peripheral blood mononuclear cells (PBMCs) for reprogramming.
Fibroblasts Peripheral blood mononuclear cells (PBMCs)
Gold standard for reprogramming efficiency and differentiation (+) Reprogramming efficiency is much lower than for fibroblasts. Can yield hiPSCs with undesirable germ lines and raises concerns about blood infections (e.g. hepatitis C, HIV) (-)
Involves an invasive biopsy of donor tissue by specialists (-) Easily isolated by routine venipuncture by a phlebotomist, with minimal risk to the donor (+)
Have to be expanded in vitro to generate sufficient quantities of cells before reprogramming can occur (-) Sufficient quantities can be extracted for immediate reprogramming, without the need for expansion in vitro (+)
Convenient cell source from blood banks, and can be extracted from patients of any age (+)

Conclusion

There are many options to consider when sourcing hiPSCs for your disease modeling research and cell therapy applications. It is clear that non-integrating reprogramming systems offer an advantage over integrating systems, as they provide high quality hiPSCs that typically do not contain any remnants of reprogramming vectors, which is a key requirement for clinical application.

Non-integrating episomal vector and Sendai-virus reprogramming technologies are reliable and efficient options for the generation of hiPSCs, and both fibroblasts and PBMCs can be re-programed using these technologies. However, each system and cell type has its own advantages and limitations in the generation of hiPSCs, and these need to be assessed to determine which is best suited to your research needs when using our custom reprogramming service.

As experts in cell reprogramming and hiPSC differentiation, we offer collaborative custom services to source a fast, reliable and convenient supply of high quality hiPSCs. All our cells originate from one from one stable donor, providing you with access to consistently standardized cells ( read our blog for further information on how hiPSCs could benefit your research). We can also differentiate and edit the hiPSCs (using CRISPR-Cas9 gene editing) to your desired final cell state and function, (e.g., neural, immune and cardiovascular cell types) (Figure 1).

Therefore, without depleting your time and resources on generating the cells you need, you can ensure the success and reproducibility of your research, and potentially change the lives of many patients around the world.

Figure 1 : Axol can directly differentiate your hiPSCs into different cell types that you need for your research.
human iPSC-derived cortical neurons

hiPSC-derived cortical neurons

human iPSC-derived cardiomyocytes

hiPSC-derived cardiomyocytes

human iPSC-derived macrophages

hipsc-derived macrophages


References

Chou, B. et al., 2011. Efficient human iPS cell derivation by a non-integrating plasmid from blood cells with unique epigenetic and gene expression signatures. Cell Research , 21, pp.518–529.

El Hokayem, J., Cukier, H. & Dykxhoorn, D., 2016. Blood Derived Induced Pluripotent Stem Cells (iPSCs): Benefits, Challenges and the Road Ahead. Journal of Alzheimer’s Disease and Parkinsonism , 6, p.275.

Kang, X. et al., 2015. Effects of Integrating and Non-Integrating Reprogramming Methods on Copy Number Variation and Genomic Stability of Human Induced Pluripotent Stem Cells. PLOS One , 10, p.e0131128.

Li, J. et al., 2014. Advances in understanding the cell types and approaches used for generating induced pluripotent stem cells. Journal of Hematology & Oncology , 7, p.50.

Malik, N. & Rao, M., 2013. A Review of the Methods for Human iPSC Derivation. In U. Lakshmipathy & M. Vemuri, eds. Pluripotent Stem Cells: Methods and Protocols . Humana Press, pp. 23–33.

Okita, K. et al., 2011. A more efficient method to generate integration-free human iPS cells. Nature Methods , 8, pp.409–412.

Schlaeger, T. et al., 2015. A comparison of non-integrating reprogramming methods. Nature Biotechnology , 33, pp.58–65.

Scudellari, M., 2016. How iPS cells changed the world. Nature , 534, pp.310–312.

Takahashi, K. et al., 2007. Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors. Cell , 131, pp.861–872.

Táncos, Z. et al., 2016. Establishment of induced pluripotent stem cell (iPSC) line from a 75-year old patient with late onset Alzheimer’s disease (LOAD). Stem Cell Research , 17, pp.81–83.

Yu, J. et al., 2007. Induced pluripotent stem cell lines derived from human somatic cells. Science , 318, pp.1917–1920.


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