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.
For all the excitement surrounding induced pluripotent stem cells (iPSCs), there has been one nagging fly in the ointment. Though the cells appear to be the functional equivalent of embryonic stem cells (ESCs), some studies have suggested they are not. But now those fears seem to have been put to rest.
As Peter Park of Harvard Medical School, Konrad Hochedlinger of Massachusetts General Hospital and colleagues reported recently in Nature Biotechnology, human iPSCs and ESCs are “molecularly and functionally equivalent” [1]. Differences in iPSC and ESC lines, they concluded, likely result mostly from differences in genetic background, rather than how they were created in the lab.
That’s good news for researchers, many of whom are exploiting these cells for applications in basic research, disease etiology and therapeutics. If you want to join that group, there’s a wide array of tools to help. Here are some of the latest offerings.
Making iPS cells
iPSCs are basically embryonic stem cells without the controversy—adult somatic cells (such as fibroblasts) that are “reprogrammed” to an embryonic-like state by the expression of four or five key transcription factors. Researchers have devised several options for introducing those factors, each with its own pros and cons.
Lentiviral vectors, such as the doxycycline-inducible Stemgent® Reprogramming Lentiviruses available from Sigma-Aldrich, are one popular choice. But as these viruses integrate into the host genome, some researchers—especially those pursuing therapeutic applications—tend to shy away from such strategies. “Making iPS cells that are vector-free”—that is, without cellular genetic modification—“is becoming more and more of a need,” says Dennis Clegg, professor and codirector of the Center for Stem Cell Biology and Engineering at the University of California, Santa Barbara.
One alternative uses DNA plasmids to drive protein expression. Cellular Dynamics Inc. (CDI), a company that specializes in the high-throughput generation and banking of iPS cells and of the terminally differentiated cells created from them, uses that strategy, says Chris Parker, the company’s chief business officer. Researchers can implement a similar strategy in their own labs using Thermo Fisher Scientific’s Epi5™ Episomal iPSC Reprogramming Kit.
Other nonintegrative options are RNA-based, including systems based on Sendai (RNA) virus (e.g., Thermo Fisher Scientific’s CytoTune-iPS 2.0 Sendai Reprogramming Kit) and modified mRNAs (e.g., Stemgent’s mRNA Reprogramming Kit). These kits typically include modified RNAs and/or the B18R protein to reduce cytotoxic responses to the transfected RNA, states Brad Hamilton, senior director of science and business development at Stemgent.
Recently, kits based on “self-replicating” RNAs have become available. EMD Millipore was the first to market with its Simplicon™ Reprogramming Kit, winner this month of an R&D 100 Award in the analytical/test category, but similar kits are now also available from Stemgent (StemRNA™-SR Reprogramming Kit) and STEMCELL Technologies (ReproRNA™-OKSGM).
Though based on the same underlying vector, these self-replicating systems differ in the reprogramming factors they express. Simplicon, for instance, expresses the OKS factors (Oct4, Klf4, Sox2) plus Glis1, whereas StemRNA-SR encodes OKS plus c-Myc. ReproRNA-OKSGM encodes all five factors: OKS, c-Myc and Glis1.
Any of these methods can, in theory, reprogram any somatic cell type. In practice, though, that’s not the case. Blood cells, for instance, are particularly problematic, says Nick Asbrock, stem cell and molecular biology product manager at EMD Millipore. Blood represents an easy source of material for reprogramming, and researchers obviously are keen to use it. But not all blood cells are equally amenable: “The types of blood cells most people want to reprogram are PBMCs [peripheral blood mononuclear cells],” Asbrock explains—that is, nucleated cells, such as lymphocytes.
Sendai virus and episomal strategies can reprogram directly from PBMCs, but Simplicon cannot yet do so, Asbrock says. Neither can StemRNA-SR, though Stemgent does offer a protocol for reprogramming from blood endothelial progenitor cells (EPCs), a step that adds two to three weeks, Hamilton says.
iPS cell media
After you’ve created iPS cells, you’ll need to maintain them in culture. That requires specialty media, says Clegg, noting that “iPS cells are a little more challenging than the typical mammalian cell lines.”
Clegg, for instance, typically grows his iPS cells in media called mTeSR™1, from STEMCELL Technologies. “We liken mTeSR1 to a diet of McDonald’s hamburgers,” he quips—the media is rich, and cells thrive on it.
There are other options, such as Essential-8, a formulation developed by stem cell pioneer Jamie Thomson. Thermo Fisher Scientific recently launched a new version of this medium, called Essential-8 Flex, which affords researchers some flexibility in their cell culture scheduling, says Uma Lakshmipathy, the company’s director of research and development for cell biology.
“If you look at pluripotent stem cell culture, it’s very unforgiving. You have to come in every day,” Lakshmipathy explains. Essential-8 Flex is formulated to last for a longer time, for instance by using a more stable form of the growth factor FGF, so media needn’t be changed quite so frequently. “You can alternate days, or not come in on the weekend,” she says.
Stemgent’s parent company ReproCell has launched another useful medium, ReproNaïve, which is capable of converting iPS cells into so-called naïve stem cells. According to Hamilton, human pluripotent stem cells are not quite as flexible as their murine counterparts. For instance, mouse embryonic stem cells can grow into three-dimensional structures, whereas human ESCs grow in a monolayer. Also, murine cells can be single-cell passaged for rapid culture expansion; human cells require the use of a ROCK inhibitor.
ReproNaïve effectively converts human ES or iPS cells into naïve stem cells, thereby imparting those features. “It’s a nice tool for disease modeling, drug screening and ultimately, cellular therapy,” Hamilton says. (He adds that researchers can effect the same change using a combination of small molecules available exclusively from Stemgent, as described by the Jaenisch lab in 2014 [2].)
Differentiation tools
Of course, for most researchers, the point of iPS cells isn’t to maintain them, but to differentiate them into something else. “iPSCs are a means to an end,” says Parker. Several companies have released reagents to aid in that process.
Thermo Fisher Scientific, for instance, launched its PSC Definitive Endoderm Induction Kit, says Lakshmipathy—a two-day protocol to differentiate cells into endoderm, the germ layer from which liver and pancreatic cells, among others, arise.
Similarly, Bio-Techne launched its StemXVivo Hepatocyte Differentiation Kit, a system of media and reagents that can, in about 20 days, convert iPS cells into liver cells—one of two key cell types for toxicity testing, says Joy Aho, the company’s stem cell product development manager.
To monitor the progress of differentiation protocols, researchers typically use qPCR or fluorescence-based techniques. Clegg, who studies diseases of the eye, tends to grow a relatively small number of cells (about 100,000) in relatively compact structures—the better for implantation in a confined space. Thus, he can study them via immunofluorescence and automated microscopy, looking for the expression of ocular markers.
EMD Millipore’s SmartFlares offer an alternative approach. A SmartFlare, explains Don Weldon, lead application development scientist for RNA detection, is a sequence specific method for identifying RNA in intact cells, “a live-cell biomarker detection tool.” SmartFlares aregold nanoparticle conjugated to oligonucleotides complementary to an RNA of interest. Those oligos are hybridized to a second short oligonucleotide, which is coupled to a fluorophore. Initially, that fluorescence is quenched by the proximity of the gold. But when introduced into cells, these particles bind their cognate RNA, releasing the short probe and inducing a fluorescent signal.
He adds that EMD Millipore has recently optimized the SmartFlare protocol to make these probes compatible with pluripotent stem cells. And the company has developed reagents for many of the common pluripotency markers, including nanog, Sox2 and Klf4, as well as some “1,500 targets for specific cell types and events.”
If your lab lacks the expertise or time to tackle the subtleties of stem cell culture, you can always outsource.
CDI offers a dozen pre-made terminally differentiated cell types, made from healthy iPS cells, under its iCell™ brand. These products include cardiomyocytes, hepatocytes and neurons, all of which are useful for drug development and toxicity testing, Parker says. Alternatively, researchers can contract with the company to create and differentiate iPS cells using the researchers’ own blood or fibroblast samples—an offering called MyCell™ Products. CDI can even genetically modify those cells, if desired, to introduce or correct specific gene mutations.
“What MyCell does is allow us to introduce disease biology,” Parker explains. “That moves us from safety and toxicity testing to discovery.”
Indeed, with such a rich and growing toolkit in the iPS cell space, those discoveries have never been closer.
References
[1] Choi, J, et al., “A comparison of genetically matched cell lines reveals the equivalence of human iPSCs and ESCs,” Nat Biotechnol, 33:1173-81, 2015. [PMID: 26501951]
[2] Theunissen, TW, et al., “Systematic identification of culture conditions for induction and maintenance of naive human pluripotency,” Cell Stem Cell, 15:471-87, 2014. [PMID: 25090446]