Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
Even more astonishing than our ability to induce mature adult cells to revert to a pluripotent state – in which they could hypothetically become any type of cell – is the fact that there are already a variety of methods to accomplish such a task. By forcing a somatic cell to express four particular genes, or factors (sometimes called ‘Yamanaka factors’ after the method’s originator), the cell can be “reprogrammed” to become pluripotent once again. Reprogramming fibroblasts to become induced pluripotent stem (iPS) cells won Shinya Yamanaka the 2012 Nobel Prize in Physiology or Medicine, which he shared with Sir John Gurdon of Cambridge University for his landmark cloning research in 1962. “Reprogramming is an incredibly dynamic field,” says Mark Powers, research and development leader at Life Technologies. “It has been less than a decade since the first human cells were successfully reprogrammed, and in that time, the pace of innovation has been phenomenal.” Now only a few years later, researchers have a number of different reprogramming methods at their disposal.
Reprogramming with viruses
Yamanaka’s seminal stem cell reprogramming research used an adenovirus-based vector to introduce Yamanaka factors or genes into the target cells. But adenovirus, it turns out, is a relatively inefficient method of generating iPS cells, so researchers who want to use a viral vector might consider the more efficient retroviruses or lentiviruses. “Lentivirus is currently the most efficient method for reprogramming, as the lentivirus inserts the reprogramming genes randomly into the host genome, forcing expression of the Yamanaka reprogramming factors,” says Nick Asbrock, product manager in stem cell and cell biology at EMD Millipore. “Lentiviral-based systems are very reproducible, and are effective in reprogramming even the hardest-to-reprogram somatic cell.” Their high efficiency also makes them relatively inexpensive.
Greater efficiency comes at a price, though: integration into the host cell’s genome, which can be problematic for subsequent experiments planned for the iPS cells. “The genes – many of which are oncogenes – are permanently inserted into the host genome,” says Asbrock. “Many researchers, especially ones looking to use iPS cells or their derivatives in the clinic, do not want these viral genes in their cells for obvious reasons.”
EMD Millipore’s lentiviral-based stem cell reprogramming system, STEMCCA, gives high reprogramming efficiencies with the option to excise the four Yamanaka factors out of the genome after iPS cell generation. “This technology is like having the best of both worlds – high efficiency and non-integrative capabilities,” says Asbrock. “We are launching a product within the next few months, a fusion protein termed TAT-Cre Recombinase, that makes the excision of STEMCCA up to 75% efficient.”
Reprogramming with episomal DNA
Reprogramming by delivering episomal DNA for the expression of the Yamanaka factors is a nearly integration-free means of generating iPS cells. Episomal DNA vectors are virus-free, safe to use, stable, and inexpensive. Generally, only a small number of transfections are necessary, and the chance of integration into the host cell genome is slight.
A disadvantage of the episomal DNA method is that it tends to be inefficient (compared to using lentiviral vectors, for example). To increase efficiency, you can use a mixture of episomal DNA vectors for reprogramming. For example, Powers says that Life Technologies’ Episomal iPSC Reprogramming Vectors are a combination of “three vectors with the oriP/EBNA-1 (Epstein-Barr nuclear antigen-1) backbone, delivering six reprogramming factors (Oct4, Sox2, Nanog, Lin28, Klf4 and L-Myc) in addition to SV40LT. These are introduced into cells via electroporation, and replicate by binding of multiple EBNA-1 homodimers to oriP within the nucleus.”
The chance of genomic integration using episomal DNA is small but not zero. If you are making iPS cell lines, you can verify that no disruption of the host cell genome occurred by repeatedly passaging and screening the cells, though this requires extra time and incurs extra costs.
Reprogramming with Sendai virus or mRNA
Some types of research require iPS cells that have been reprogrammed with zero chance of genomic integration, such as clinical studies or therapeutic applications (see below). One reprogramming method that is free of genetic perturbation uses vectors derived from Sendai virus, an RNA-based virus that is non-integrating and replication-deficient. “Sendai virus acts exclusively in the cytoplasm of cells and cannot enter the nucleus or alter host chromosomes,” says Powers. “It is therefore fundamentally free of the risks associated with conventional viral vectors.” Life Technologies’ CytoTune®-iPS Sendai Reprogramming Kit includes Sendai viral vectors with four transcription factors (Oct4, Sox2, Klf4, and c-Myc) to reprogram somatic cells. Sendai viral vectors also reprogram cells with relatively high efficiency.
Disadvantages of using Sendai virus include relatively higher costs compared to other methods (though the higher efficiency means less transductions required), as well as the inconvenience of higher biosafety containment measures. Also, if you need to prove the absence of viral material in your iPS cell lines, you may be in for a lot of work. “Establishment of virus-free iPS cell lines requires 10 to 20 passages, depending on the cell line and reprogramming conditions, and significant molecular screening,” says Brad Hamilton, R&D director at Stemgent. “You must isolate and clonally propagate at least 10 primary colonies to establish only a few iPS cell lines per patient sample.”
However, the Sendai virus system is ideal for reprogramming blood cells, an area of growing interest due to the ease of obtaining these samples from patients. Additionally, and there are many epidemiological studies that have large banks of blood samples in storage that can now be reprogrammed with Sendai virus – where mRNA has not been shown to efficiently reprogram (hematopoietic) blood cells.
Another integration-free reprogramming method is mRNA delivery. The ability to reprogram stem cells using a method that is both non-integrative and non-viral is especially important for future therapeutic applications. “The mRNA reprogramming system is ideally suited for the generation of human iPS cell lines in that the resulting lines are completely free of viral programs or unwanted genomic integrations that may ultimately affect the cell line’s subsequent utility in disease modeling, screening, and other therapeutic applications,” says Hamilton. Stemgent is currently focusing on mRNA-based methods to generate human iPS cell lines from both fibroblasts and various blood cell types. “The advantage of this technique is that there is no risk for insertional mutagenesis that can later cause cancer,” agrees Anton McCaffrey, department head of cell biology at TriLink Biotechnologies, which specializes in the production of mRNAs for stem cell reprogramming and gene therapy application. “For cells that will eventually be used in the clinic, this will likely be the dominant technology. This is also true in the gene therapy field.”
Besides the lack of genomic disruption, other advantages of this method include speed and high efficiency. For example, mRNA reprogramming can result in iPS colonies within 2 weeks, and according to Hamilton, can be “as high as 10% when starting with 5,000 target fibroblasts.” Also, mRNA reprogramming has a “high success rate of over 90% of iPS cell line establishment after primary colony isolation,” he says. “[There’s] no need to isolate and maintain a large number of colonies to establish only a few cell lines.”
One disadvantage of mRNA reprogramming is the inconvenience posed by the daily mRNA transfections required for this method. Another disadvantage is that it only works with the Pluriton medium, which makes this system less flexible and adds to the already high cost of the kit. But unlike other methods, there is no need to screen the resulting clones (in order to weed out colonies containing viral components or integrated oncogenes, for example). Thus, even though the mRNA method is more expensive up front, the total costs across methods may be closer than they appear at first. “Ultimately, the mRNA method dramatically reduces derivation costs per iPS cell line generated,” says Hamilton, “as there is no need for the laborious and time-consuming screening of isolated clones that is necessary when reprogramming with both the Sendai virus and episomal DNA systems.”
Animal-free iPS cells
Early systems for generating iPS cells, notes StemCell Technologies’ scientist Arwen Hunter, used mouse embryonic feeder cells and undefined growth media. Not only do these methods give inconsistent results, but they also rule out clinical use in humans. With new media available today, iPS cell lines derived from mouse embryonic feeder systems “can be adapted to feeder-free growth conditions,” she says. Today, the generation of iPS cells without feeder cells, under defined-media conditions, advanced the technology closer toward the clinic. “StemCell Technologies provides TeSR™-E8™, Vitronectin-XF™ and to support xeno-free, defined reprogramming methods,” says Hunter. Indeed, clinical applications of iPS cell lines entail making sure that all reprogramming methods and materials are GMP-compliant, so that cell lineages are documented and completely free of non-human, animal materials. Stemgent is also facilitating this process with their xeno-free Pluriton™ Reprogramming Medium, which along with “our miRNA cocktail creates a unique platform for reprogramming,” says Hamilton, “where the reagents are xeno-free, defined and capable of efficiently generating truly integration-free iPS cell lines in under two weeks from the most difficult-to-reprogram patient fibroblast samples.”
Life Technologies also offers Essential 8 Medium, a fully defined, xeno free system for the culture of PSCs. Essential 6 Medium, which was just launched in March, is ideal for EB formation and is an important component of the xeno free reprogramming and differentiation processes. Both of these products, developed in Dr. Jamie Thomson’s laboratory, are manufactured under cGMP conditions giving customers full confidence in the consistency and reproducibility of their results. Life also offers the only cell therapy ready products with our CTS™ branded systems that we expect to play a significant role in the transition from research to clinical applications.
Challenging samples and stubborn cells
Despite the multiple reprogramming methods at their disposal, researchers are still dogged by two challenges: sample variability, and cell types that are resistant to reprogramming. “Variability in the tissue harvesting, cell expansion, and cryopreservation techniques utilized in research labs ultimately results in target cell samples that have varied age/passage numbers, proliferation rates, and viability,” says Hamilton. The resulting variations in target cells can make it difficult to derive iPS cells from the required samples. Stemgent focuses on designing methods that will be effective for even difficult-to-reprogram cells. “By incorporating a cocktail of miRNAs into our mRNA-based reprogramming system, we have been able to generate iPS cell lines from target cells that have been previously resistant to reprogramming with various viral reprogramming methods, including Sendai virus,” says Hamilton.
Powers agrees that clinical samples pose substantial hurdles. “The most significant challenges faced by researchers today revolve around the difficulties in reprogramming a broad array of patient-derived somatic cells – most notably blood cell types,” he says. “Many systems simply do not reprogram these cells well, if at all.” Life Technologies designed their episomal DNA and Sensai virus reprogramming methods to be effective in generating iPC cells from blood cells. “For these reasons many researchers and institutions have standardized methods around these technologies in creating patient derived iPSC,” says Powers. “Continual effort is focused on improving these and other methods through further optimization of vector design, the use of additional reagents and protocol optimization for increased efficiency, reduced effort, and minimization of cost.”
The 2012 Nobel Prize marked the beginning of stem cell research applications in medicine. If news of the recent licensing of Moderna Therapeutics’ mRNA reprogramming technology by AstraZeneca for $240 million is any indication, the race to bring stem cell technology to patients’ bedsides is in full swing.