by Caitlin Smith
Stem cell researchers are getting excited, and with good reason. Methods are emerging to reprogram cells from one type to another and to reprogram adult cells (for example, fibroblasts) into induced pluripotent stem cells (iPSCs)—a state much like embryonic stem (ES) cells—from which they can become any cell type. This is especially important for researchers, because they may be able to use iPSCs instead of the more controversial embryonic stem cells.
Although it is unknown whether iPSCs and embryonic stem cells are clinically equivalent, iPSCs are already being used in disease modeling and drug development. One day, iPSCs may be used to create the needed adult cell types for tissue transplantation. “The ability to directly transform cells from one lineage to another has huge implications for both basic biology as well as the clinic,” says Clive Glover, senior product manager at StemCell Technologies. “At this point, the field [of reprogramming] is very new. More work needs to be done to uncover the mechanisms behind these observations.” Other challenges that need to be overcome include reprogramming efficiency, clinical safety, speed of experiments and unintended effects of reprogramming.
Current trends in stem cell reprogramming
Earlier work on iPSC reprogramming was performed in the lab of Shinya Yamanaka, professor of developmental engineering and director of the Center for iPS Cell Research and Application at Kyoto University, using co-transduction of four retroviruses. However, a newer trend is reprogramming using small RNA molecules. “The most exciting development in the field of stem cell reprogramming would be the new reprogramming method that does not require vector-based gene transfer, such as reprogramming using synthetic mRNA of transcription factors and reprogramming using mature microRNA,” says Danqiong Sun, associate scientist, Stem Cell Department at System Biosciences (SBI). “We are planning to offer those new re-programming tools in the near future. Both mRNA reprogramming and mature microRNA reprogramming have shown higher than average efficiency. And those new technologies involve neither virus infection nor genetic integration. They represent safe and efficient strategies for iPSC generation, and they hold the potential for biomedical research and regenerative medicine. Patient-specific iPSCs generated with ‘safe’ technologies from patient somatic cells can potentially be used for cell therapy. A combination of patient-specific iPSCs and gene correction represents a powerful tool of gene and cell therapy in the future.”
Another group headed by James Eberwine recently used mRNAs to reprogram adult cells, showing “direct reprogramming of somatic cells into other lineages, which demonstrates the conversion of both fibroblasts and astrocytes into cardiomyocytes using mRNAs,” [1] says David Welch, senior market development manager for primary and stem cell systems at Life Technologies. “This exciting area of reprogramming research not only holds promise for therapeutic applications but also provides insight into the mechanisms of reprogramming.” Reprogramming adult cells to become iPSCs rather than relying on rare embryonic stem cells would help to avoid the ethical issues surrounding use of the latter.
But Yang Xu, professor of molecular biology at the University of California, San Diego, who uses an episomal approach in his lab to create iPSCs, urges caution. “All existing reprogramming technologies remain problematic because they all induce genetic and epigenetic abnormalities,” says Xu. “We need to optimize the reprogramming approaches to minimize the genomic and epigenetic abnormalities, to achieve the potential of iPSCs in human therapy and disease modeling.” Recent work from Xu’s lab showed a surprising result: In mice, the immune system of an individual mouse rejected iPSCs from that same mouse but tolerated embryonic (ES) cells from different mice.[2] Xu suggests that we exercise caution when using adult-derived iPSCs for clinical applications—changes in gene expression may occur during reprogramming, attracting the attention of the immune system in a way embryonic stem cells do not.
Current tools
Cell Biolabs
Many tools today are aimed at improving reprogramming efficiency. For example, using four retroviruses to reprogram requires integration from four different vectors, which can reduce efficiency. “We offer the pLentG-KOSM Lentiviral Plasmid, which contains all four genes in one vector,” says Ken Rosser, director of business development at Cell Biolabs. “This not only simplifies the workflow, since only one virus needs to be made, but it also provides on average 10-fold higher efficiency of reprogramming compared to the multi-virus approach and much higher [efficiency] than non-viral methods.”
System Biosciences (SBI)
SBI also offers new tools to smooth the reprogramming pathway. “We have recently launched microRNA cluster miR302bcad/367 in both lentivector/lentivirus and minicircle formats for reprogramming,” says Sun. “As published [in] Cell Stem Cell, Anokye-Danso, et al. showed that the miR302/367 cluster rapidly and efficiently reprograms mouse and human somatic cells to iPSCs without exogenous transcription factors. The expression of miR302/367 produced two orders of magnitude more iPSC clones than when the traditional four transcription factors were used.[3] In additional to the integrative lentivirus method, we added minicircle format for microRNA expression without genomic integration and non-viral iPSC generation.”
StemCell Technologies
Additionally, StemCell Technologies offers its STEMcircles—a non-viral, non-integrating tool for reprogramming somatic cells into iPSCs. “Unlike conventional DNA plasmids, the STEMcircles™ minicircle vector contains no bacterial DNA and can therefore evade silencing mechanisms that cells naturally use against foreign DNA,” says Glover. “The result is more robust and prolonged gene expression and superior reprogramming over that of regular DNA plasmids.” StemCell Technologies also offers a special type of feeder-independent expansion medium for human pluripotent stem cells, called mTeSR1. “It has been shown to support the derivation of human-induced pluripotent stem cells without the use of feeders,” says Glover. “This means that reprogramming can be done in a more defined environment, which is advantageous if the cells are to be used for clinical purposes.” Life Technologies also offers its KnockOut™ ESC/iPSC Media Kit for feeder-based expansion, StemPro® hESC SFM for feeder-free iPSC expansion and KnockOut™ SR XenoFree CTS™ for clinical applications.
Life Technologies
Life Technologies recently launched the CytoTune™-iPS Reprogramming Kit, which uses the Sendai virus as a tool to reprogram somatic cells into iPSCs. Use of the Sendai virus has two advantages over other viral types. Being an RNA virus, Sendai does not integrate into the host genome (as can lentivirus, retrovirus or adenovirus), which can inadvertently disrupt gene expression. In addition, the Sendai virus gives the kit a 100-fold higher efficiency than standard viral methods. “The development of novel technologies such as Sendai virus, mRNA and miRNA allow more efficient reprogramming without integration,” says Welch. “This allows reprogramming and subsequent selection of iPSCs to become less of a bottleneck, and the resulting integration-free iPSCs [to] have a broader range of use, particularly in screening and cell therapy applications.”
The inefficiency of standard viral methods results in many non-transformed and partially reprogrammed cells, making it difficult to select pluripotent colonies. “At this year’s International Society for Stem Cell Research (ISSCR) meeting, we presented work on a novel live alkaline phosphatase stain which can be used to identify iPSCs without sacrificing the stained cells,” says Welch. “This allows researchers to rapidly and easily identify the putative iPSCs colonies and continue expanding them for banking and differentiation.”
Stemgent
Stemgent offers an mRNA Reprogramming System that enables one to generate iPSCs with no vector integration. “Starting with the launch of our mRNA Transcription Factors Set, we used the same substituted nucleotides and CAP analog in our in vitro transcription reaction as reported by Warren et al. 2010 to reduce eliciting an interferon response in cells subjected to repeated transfections with exogenous mRNA,” says James Kehler, product manager at Stemgent. “In addition, we developed a synthetic lipid Stemfect mRNA Transfection Reagent to deliver mRNA with exceedingly high efficiency (95-99%) and low toxicity in a variety of human cell types.” They also offer a new xeno-free Pluriton™ mRNA Reprogramming Medium, which was specifically designed for mRNA reprogramming. “While our understanding and manipulation of pluripotency in mouse cells has matured over the last 30 years into a codified technology facilitating our ability to perform functional genetics, human pluripotential cell culture is still in its adolescence,” says Kehler, who adds that Stemgent’s goal is to make the generation of iPSCs a more reliable and reproducible method for researchers.
EMD Millipore
More tools for successful reprogramming are also offered by EMD Millipore, who recently released a humanized version of their STEMCCA;trade; technology in the form of their Human STEMCCA Cre-Excisable Constitutive Polycistronic (OKSM) Lentivirus Reprogramming Kit. “STEMCCA is a polycistronic lentiviral vector that expresses the Yamanaka factors for reprogramming somatic cells into iPS cells,” says Amy Noble, marketing product manager for stem cells at EMD Millipore. “This humanized version is more efficient than the original mouse version.” Another helpful tool is their Human iPS Cell Boost Supplement, a small molecule supplement that you add to your reprogramming media. “For human reprogramming, it significantly increases the efficiency when used in conjunction with STEMCCA, as well as the quality of your iPS cells, and decreases the time it takes to generate your iPS cells,” says Noble. “You get a nice morphology that is more like the human ES cell morphology, cells are easier to passage, and it cuts the time by half (from about 60 to 30 days).” The combination of small molecules in the supplement is proprietary, but contains factors that affect signaling pathways known to be important for reprogramming.
One of the biggest initial hurdles in reprogramming that researchers may encounter is whether they have previous ES cell experience, says Vi Chu, stem cell R&D manager at EMD Millipore. “It takes a trained eye to be able to recognize cells that are truly pluripotent, versus those that are more differentiated,” says Chu. “To that end, we do have a kit that helps researchers to quickly identify which of their reprogrammed cells are truly reprogrammed, versus partially reprogrammed. Our Human iPS Cell Selection Kit contains a collection of conjugated antibodies that target specific pluripotent markers. Only in the truly reprogrammed human iPS cells would both positive markers for pluripotency show up.” Another challenge is characterizing the fully reprogrammed cells, “including whether or not the reprogramming transgenes have been silenced. Full reprogramming should silence the transgenes, and the exogenous pluripotency circuitry should be activated and would allow the cells to continue in an ES-cell-like state,” says Chu. EMD Millipore’s STEMCCA Viral Gene Detection qPCR Multiplex Kit is designed to help with this – it is a real-time PCR-based kit that lets you monitor in real time whether the transgenes are effectively silenced, using their proprietary Amplifluor® technology.
Current limitations and future developments
The biggest limitations today in stem cell reprogramming include low efficiency, slow time line of reprogramming experiments and safety. “The low efficiencies that are typical of most reprogramming experiments and the length of time that these experiments take really slows down the progress that could be made in this field,” says Glover. “As more understanding is gained around the mechanisms behind reprogramming, it is inevitable that these impediments will be overcome.”
Safety concerns must be addressed before reprogrammed stem cells can be used in clinical applications. “It has been shown in mouse models recently that iPSCs can induce T-cell-dependent immune responses in syngeneic recipients,” says Sun. “Safer and more efficient reprogramming methods, such as mRNA reprogramming, have been developed to overcome the low efficiency, slow kinetics and safety issues. For immunogenicity, patient-specific iPSCs should be evaluated before any [clinical] application of these autologous cells into the patients.”
Another obstacle is agreeing on criteria for characterizing iPSCs. “There is still a lack of consensus in the research community on how to prove that a putative iPSC is truly pluripotent,” says Welch. “Shinya Yamanaka presented some interesting work at this year’s ISSCR demonstrating that the teratoma assay is not always reliable as a standard test for pluripotency, and tools such as the iPSC scorecard developed by Kevin Eggan allow a more global picture of pluripotency. A scalable and modular set of characterization tools providing information on gene expression, epigenetic profile, cell identity and more will allow researchers to confidently use their reprogrammed cells in basic research, drug discovery and cell therapy applications.”
Paul Knoepfler, associate professor in cell biology and human anatomy at the University of California, Davis, wishes for a highly efficient chemical approach to reprogramming. “We are most excited about genomic and epigenomic approaches to studying reprogramming, given the importance of these areas for iPSC and embryonic stem cell function.” He explains, though, that safety remains an important concern. “From my perspective, perhaps biased by my tumor biology background, the biggest hurdle is still safety, not because we know that iPSCs are unsafe (the jury is still out on that), but rather because we know so little about their potential tumorigenicity. However, I think that is going to change in the next one to two years as scientists are hammering away at this issue. We already know that iPSCs have changes in their genomes and epigenomes, but are these changes meaningful? I think we’ll find out soon.”
References
[1] Kim TK, Sul J, Peternko NB, Lee JH, Lee M, Patel VV, Kim J, Eberwine JH. (2011) “Transcriptome transfer provides a model for understanding the phenotype of cardiomyocytes.” PNAS, July 5.
[2] Zhao T, Zhang ZN, Rong Z, Xu Y. (2011) “Immunogenicity of induced pluripotent stem cells.” Nature, 474(7350): 212-215.
[3] Anokye-Danso F, Trivedi CM, Juhr D, Gupta M, Cui Z, Tian Y, Zhang Y, Yang W, Gruber PJ, Epstein JA, Morrisey EE. (2011) “Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency.” Cell Stem Cell, 8(4): 376-388.
The image at the top of the page is from Life Technologies' CytoTune™-iPS Reprogramming Kit Brochure