Jeffrey Perkel has been a scientific writer and editor since 2000. He holds a PhD in Cell and Molecular Biology from the University of Pennsylvania, and did postdoctoral work at the University of Pennsylvania and at Harvard Medical School.
It’s only been 10 months since we last reviewed reagents for creating and working with induced pluripotent stem (iPS) cells. Yet the field remains as hot as ever, and developers continue to innovate. Here, we round up some of the latest tools and trends.
Self-replicating RNA
iPS cells are created by expressing four transcription factors in somatic cells, which together serve to “rewind” the cellular clock, creating an embryonic-like pluripotent cell. Traditionally, these factors are delivered using retroviral or lentiviral vectors. But more recently, researchers have developed approaches using DNA plasmids, mRNA or even the RNA-based Sendai virus.
But all these strategies have shortcomings. Retro- and lentiviruses integrate into the host genome; Sendai virus is difficult to clear from culture; and mRNA, owing to its instability, must be delivered over and over for a period of 14 days or so to induce cellular reprogramming. Plasmids, too, are verboten for certain clinical applications because of the risk of adverse genomic modification.
"We see a general trend in the iPS cell field," says Nick Asbrock, stem cell and molecular biology product manager at EMD Millipore: "People moving away from viral-based methods to make iPS cells towards safer, nonviral methods."
In 2013, a research team led by Steve Dowdy at the University of California, San Diego, described a “self-replicative RNA” that offered the genomic safety of mRNA-based iPS cell generation without the hassle of repeated transfection [1]. Based on the Venezuelan equine encephalitis (VEE) virus, this RNA encodes both iPS-cell reprogramming factors, a selectable marker and four proteins for making more copies of the RNA itself. A single transfection is sufficient to induce iPS cells from both newborn and aged somatic cells, the authors found. To eliminate the RNA, simply remove the protein B18R, which suppresses interferon signaling and results in degradation of the self-replicating molecule, from the medium.
“RNA-based methods provide a more controlled way of reprogramming, leaving the host genome untouched,” explains Asbrock, whose company, EMD Millipore, has commercialized the self-replicative RNA technology under the name Simplicon™.
Stemgent has also licensed the technology, and will launch its reagent offering this fall, says Brad Hamilton, the company’s director of research and development.
Stemgent demonstrated this past June at the International Society for Stem Cell Research (ISSCR) meeting that self-replicating RNA can be used (in combination with Stemgent's microRNA Booster Kit) to reprogram blood cells, a desirable, but traditionally difficult target for iPS cell generation.
Indeed, the need for tools to reprogram blood cells into iPS cells is growing, says Simon Hilcove, product manager for pluripotent stem cell biology at STEMCELL Technologies. "The clearest trend I see is a shift towards reprogramming of blood cell types and away from fibroblasts," Hilcove says. "Blood is a more readily available cell source either from patients, or from vast stores of samples in blood banks. Additionally, blood cells are considered less likely to have genetic abnormalities as they haven't been exposed to the same UV (sunlight) that skin-derived fibroblasts have."
“What we presented at ISSCR was the first work we know of where cells were isolated from human blood and reprogrammed with RNA technology,” Hamilton says. Stemgent currently offers blood cell-based reprogramming as a service.
Xenobiotic-free media
Thermo Fisher Scientific is set to expand its cell-culture portfolio next month with CTS™ Essential 8™ Medium, a completely chemically defined pluripotent stem cell medium for cell-therapy research applications, says Mohan Vemuri, the company’s R&D director for stem cell biology.
CTS Essential 8 Medium is a variant of Essential 8, a medium formulated for feeder-free culture of pluripotent stem cells. “Essential 8 has some human serum-derived components,” Vemuri explains. “They are now completely replaced with recombinant proteins in the CTS Essential 8 Medium.”
Another new option is EMD Millipore’s PluriSTEM-XF™ Human ES/iPS Medium. According to Asbrock, researchers can couple that medium with PluriSTEM-XF Recombinant Vitronectin, an extracellular-matrix protein, for growth in the absence of murine embryonic fibroblasts (MEFs) or tumor extracts such as BD Matrigel.
Such so-called "xenobiotic-free" formulations are particularly useful for preclinical and translational applications, Asbrock says, as animal-derived and chemically undefined products can pose regulatory hassles—not to mention the risk of unidentified pathogens.
Differentiation tools
Several companies have developed tools to direct differentiation of iPS cells into specified lineages.
The StemXVivo™ Cardiomyocyte Differentiation Kit from R&D Systems, a Bio-Techne brand, for instance, “is an all-in-one package that contains optimized reagents to reproducibly differentiate human pluripotent stem cells into cardiomyocytes in a quick and easy way,” says Julia Hatler, product manager for stem cells and custom services at R&D Systems. Released in September, the kit includes both media and a cardiomyocyte-specific antibody to monitor differentiation status. “You can see contracting cells as early as 11 days upon using this protocol and reagent set,” Hatler says.
R&D Systems also offers StemXVivo-branded kits for differentiation into endoderm, ectoderm and mesoderm, Hatler says, as well as a Human Pluripotent Stem Cell Functional Identification Kit, intended as a straightforward, fast and inexpensive method for evaluating pluripotency.
“We provide media supplements to drive differentiation into the three lineages and antibodies to characterize each of the germ layer cell types, and it only takes five days to get through the entire experiment,” Hatler notes. By comparison, teratoma assays are time consuming, labor intensive and expensive. “This isn’t a replacement but provides a valuable quality check, for instance as a pre-screen prior to conducting the more arduous teratoma assay,” she says.
Another differentiation tool is STEMCELL Technologies’ ReproTeSR, set to launch next month. Designed for “optimal reprogramming of blood-cell types” and "similar" to the company's TeSR™-E7™, ReproTeSR "is xeno-free and also yields primary iPS cell colonies with excellent morphology, so they're much easier to identify and pick,” Hilcove says.
Thermo Fisher Scientific has also launched new differentiation media. The company’s Neural Induction Medium, for instance, can expand 1 million iPS cells into 20 million to 40 million neural stem cells in seven days, Vemuri says. And these cells are highly plastic, he adds, capable of generating midbrain, hindbrain and front-brain precursors—something that isn’t always possible via more traditional methods.
More recently, Thermo Fisher introduced media for differentiating iPS cells into cardiomyocytes, which can produce a high percentage of synchronously beating cardiomyocytes from human embryonic or iPS cells in about 10 days.
Of course, for all that these new tools offer, unmet needs remain. Researchers constantly ask for reagents to reliably differentiate iPS cells into functional islet cells and blood cells, for instance, says Vemuri, and Thermo and others are on the case. Don’t be surprised, then, if the iPS cell toolbox is even more crowded next year.
Reference
[1] Yoshioka, N, et al., “Efficient generation of human iPSCs by a synthetic self-replicative RNA,” Cell Stem Cell, 13:246-54, 2013. [PubMed ID: 23910086]
Image: Alkaline phosphatase staining of reprogrammed HFF fibroblasts generated using EMD Millipore’s Simplicon RNA reprogramming technology.