by Jeffrey M. Perkel
Between research advances and legal setbacks, scientists in the stem cell community are surely suffering from collective whiplash these days.
First, the good news. The field achieved a milestone in October as Geron Corp. finally initiated its Phase 1 study of GRNOPC1, an embryonic stem cell-derived treatment for spinal cord injuries that it calls "the world's first clinical trial of a human embryonic stem cell (hESC)-based therapy in man." In November, Advanced Cell Technology
received approval from the US Food and Drug Administration to initiate the second such trial, this time for a form of heritable macular degeneration. On the flip side is the ongoing legal battle, Sherley v. Sebelius, which argues that federal support of embryonic stem cell work violates the Dickey-Wicker amendment banning funding for research that destroys human embryos. The case is muddying the water, making it difficult for scientists to plan too far ahead.
At the lab bench, however, embryonic stem cell research continues undeterred, aided by an evolving and growing set of tools for the maintenance, differentiation, and characterization of these cells.
"I think it's safe to say there's a huge amount of energy in the whole stem cell field," says Clive Glover, senior product manager at StemCell Technologies. One highly energized area of stem cell research involves so-called "induced pluripotent stem cells" (iPSCs), embryonic-like cells that are derived from adult somatic cells by the introduction of a handful of transcription factors, usually Oct4, Sox2, Klf4 and c-Myc, and sometimes Nanog and Lin-28. First described in 2006 by Kyoto University researcher Shinya Yamanaka, iPSCs promise the research and clinical benefits of ESCs without the messy ethical downsides, and several commercial iPS kits are now available. The viPS™ Vector Kit from Thermo Scientific Open Biosystems, for instance, includes six pre-packaged lentiviruses expressing the four Yamanaka factors plus Nanog and Lin28, under the control of the human elongation factor-1 alpha promoter.
To truly become valuable clinical tools, though, technical hurdles must be overcome. Reprogramming factors typically are expressed using lentiviruses. But lentiviruses insert their DNA into the host cell genome, raising the possibility of inactivating, or at least altering the expression of, critical host genes – an issue that is especially important for clinical applications and one that is amplified when each factor is carried on a separate virus. The process is also woefully inefficient, with fractional to single-digit efficiencies being typical of the field. "'High efficiency' is relative when you're talking about iPSCs," says Amy Laws, product manager at Stemgent.
EMD Millipore's STEMCCA Cre-Excisable Constitutive Polycistronic Lentivirus Reprogramming Kit enables post-reprogramming removal of the inserted lentivirus via the Cre recombinase (delivered using a recombinant adenovirus); polycistronic viruses offer the advantage of only one insertion event per reprogrammed cell, instead of one per gene. The Stemgent® Dox Inducible Reprogramming Polycistronic Lentivirus (produced by Stemgent and available from Sigma-Aldrich) enables expression-on-demand of four mouse reprogramming factors via a doxycycline-inducible promoter. According to Supriya Shivakumar, functional genomics marketing manager at Sigma Life Science, Sigma is also beta-testing a next-generation reprogramming system based on a non-integrating lentivirus. "We know the technology works, it's really just a case of validating the cell lines that we are making in the iPS realm," she says.
Others are pursuing alternatives to viral reprogramming. Researchers have already demonstrated iPS cell generation using chemical agents as substitutes for key reprogramming factors [1], and using protein delivery [2]. In November, researchers at Children's Hospital Boston described the generation of iPS cells using mRNA. [3] Another option: non-viral DNA.
Expressing four reprogramming factors (Lin28, Nanog, Sox2, and Oct3/4), as well as GFP, StemCell Technologies' STEMcircles™ are so-called "mini-circles" – small circular DNAs that lack an origin of replication and thus cannot replicate. The disadvantage of this approach is the cells need to be transfected three times in the first eight days of the procedure, Glover says. But from a safety perspective, "it means there's no trace of the original factors in the cells once you have your iPS cells." STEMcircles, he adds, are "the first commercially available non-viral, non-integrating technology for generating human iPS cells."
However you go about making your iPS cells, characterization of potential colonies is key, lest you end up propagating and characterizing cells that are only semi-reprogrammed. "What frequently happens is that, when you do a reprogramming experiment, not all colonies you see are actually truly reprogrammed," says Vi Chu, R&D manager at EMD Millipore.
Generally, this characterization can be accomplished using antibodies. Antibodies are available both individually and in pre-packaged kits from a variety of companies including EMD Millipore, Thermo Fisher Scientific, Stemgent, and Sigma-Aldrich. EMD Millipore's iPS Selection Kit, for instance, is a live-cell assay using antibodies to the pluripotency markers SSEA-4 and TRA-1-60, as well as a negative control (CD13, which stains fibroblasts). Following staining, the cells can be washed to remove the antibodies and placed back into culture. "This kit really allows you to narrow down upon which clones are most likely to be the fully reprogrammed colonies," says Chu. Stemgent's Pluripotency Characterization Set uses both antibodies and alkaline phosphatase activity to assess pluripotency. "If you're making iPS cells, you do need to do this characterization part of the assay to determine if they have fully reprogrammed or not," says Laws.
Some researchers characterize ESCs and iPSCs using mRNA abundance, instead. Life Technologies offers a broad portfolio of such assays based on its TaqMan® chemistry, available both individually and in the TaqMan Array Stem Cell Pluripotency card, which measures the mRNA levels of 92 test and six control genes in parallel. EMD Millipore's STEMCCA Viral Gene Detection qPCR Multiplex Kit assesses reprogramming efficacy using just three genes: nanog (a relatively late pluripotency marker not included on the STEMCCA transgene), Oct-4 (a reprogramming factor), and GAPDH (a control). The kit, says Chu, "allows the researcher to quantify the relative levels of the pluripotency genes that are present, and also to look to see whether or not the viral transgenes have been either silenced or completely removed."
Another high-energy area for stem cell biologists involves improving culture conditions. Human ESCs traditionally are cultured atop a layer of growth-factor-secreting feeder cells, such as murine or human embryonic fibroblasts, in basal media supplemented with serum. Alternatively, cells may be cultured on substrates such as Geltrex (Life Technologies) or Matrigel (BD Biosciences), which are extracellular matrix preparations from mouse tumors, supplemented with MEF-conditioned media. But serum, feeder layers, conditioned media, and biological substrates are all potential sources of variability, as their properties can differ from batch to batch. They also are molecular black boxes that are incompatible with stem cell technology's clinical promise. Thus, the push is on to develop well-defined, xenobiotic-free culture conditions.
In May, three back-to-back publications in Nature Biotechnology described potential solutions to at least part of the problem. [4-6] Researchers at the University of Michigan, Corning Life Sciences, and the Karolinska Institute in Sweden independently reported defined growth substrates that could support the maintenance of embryonic stem cells in culture in the absence of feeder cells or ECM. "These papers represent important steps in the development of culture technologies suitable for industrial applications of hESCs," wrote Larry Couture in an accompanying News and Views article [7].
Corning commercialized its surface under the brand name Synthemax™. Other commercial growth substrates include Thermo Scientific's Nunclon Vita Stem Cell Culture Surface and Life Technologies' CELLstart™ CTS™. Nunclon Vita, says Marketing Director Mark Collins, is "specially treated to support the expansion of embryonic stem cells using our [AdvanceSTEM™] media without the need for feeder layers." CELLstart CTS is "a defined, GMP substrate containing components of human origin," according to David Welch, senior market development manager for primary and stem cell systems at Life Technologies. (Life Technologies' GIBCO CTS [Cell Therapy Systems] brand, launched in June, "provide[s] the necessary documentation and traceability that allows scientists to use those products and take them more efficiently into a clinical application," Welch says.)
Progress also continues in the development of ever more well-defined culture media and reagents. But with so many confusing, seemingly redundant terms floating around in the product literature, it's a good idea to arm yourself with a little knowledge first.
StemCell Technologies identifies "defined" media as one whose ingredients "are greater than 95% in purity," says Glover, a definition that can include highly purified proteins. "Chemically defined" is more rigorous, and refers to media whose compounds are of "known structure and very high purity, usually chemically synthesized products." A medium described as "xeno-free" can contain human products, but not products from non-human eukaryotes. "Animal product-free" is a tougher standard, as it also precludes human protein products.
"We are always aiming to get serum free, and serum-free is usually synonymous with defined," says Glover. "Once we've gotten to serum-free and defined, the next step up is to try and go xeno-free, and then the next step up from xeno-free is to go to animal component-free, and then to chemically-defined."
Commercial pluripotent stem cell-qualified media include StemCell Technologies' mTeSR (defined) and TeSR2 (defined, animal-component free); Stemgent's Stemedia™ NutriStem™ XF/FF culture medium (defined, xeno-free); Thermo's HyClone AdvanceSTEM Cell Culture Media (serum-free); EMD Millipore's HEScGRO Medium for Human ES Cell Culture (xeno-free); and Life Technologies' StemPro® hESC SFM, KnockOut™ SR, KnockOut SR XenoFree CTS, and KnockOut D-MEM CTS. Note that while most, if not all, of these media support growth in the absence of serum with the addition of supplements, some (such as AdvanceSTEM and HEScGRO) still require feeder cells.
Of course, beyond culture conditions and iPS cell reprogramming, there exists a vast universe of tools researchers may tap into to study stem cell biology. Growing cells, says Michelle Collins, global product manager for gene expression at Bio-Rad Laboratories, "is only half the battle; the rest of the battle is conducting the experiments using various methods, instruments, and reagents to get the answer that you're looking for."
That includes everything from plate imagers and gel documentation systems, to electroporators, incubators and real-time PCR systems. For example, Bio-Rad's new TC10 Automated Cell Counter fills an important stem cell niche because, Collins says, "if you over- or under-estimate your cell count, it can be detrimental to the life of your cells." On the higher end of the scale, for those researchers looking to transition their research into the clinic but who lack the cleanroom environment required to grow cells under GMP conditions, there's the Sanyo CPWS Integrated Cell Processing Workstation – essentially, a "class 10,000 cleanroom-in-a-box," says Deepak Mistry, manager for strategic development and marketing at Sanyo North America.
"Let's say you have a biotech involved in stem cell research and they're not at that stage where they're going to go into full complete production, but they are at the point where they are beginning trials," Mistry says. "This could be a great product for them to immediately get the work that they need to get done for proof-of-concept."
Despite the breadth of tools available to the stem cell community, unmet needs do remain, including developing consistent, well-defined culture systems and protocols for easy comparison of results between labs; establishing conditions for pluripotent cell differentiation down defined lineages; and improving the efficiency of iPS cell reprogramming. Work continues on these fronts.
In the meantime, at least when it comes to iPS cell generation, help is available. According to Laws, Stemgent recently opened a hands-on training program in San Diego to teach the fundamentals of reprogramming and iPS cell generation. "Right now," she says, "the field is just so young, and rapidly evolving."
References
[1] B. Feng et al., "Molecules that promote or enhance reprogramming of somatic cells to induced pluripotent stem cells," Cell Stem Cell, 4[4]:301-12, 2009.
[2] H. Zhou et al., "Generation of induced pluripotent stem cells using recombinant proteins," Cell Stem Cell, 4[5]:381-4, 2009.
[3] L. Warren et al., "Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA," Cell Stem Cell, 7[5]:618-30, November 2010.
[4] L.G. Villa-Diaz et al., "Synthetic polymer coatings for long-term growth of human embryonic stem cells," Nat Biotechnol, published online 30 May 2010, doi:10.1038/nbt.1631.
[5] Z. Melkoumian et al., "Synthetic peptide-acrylate surfaces for long-term self-renewal and cardiomyocyte differentiation of human embryonic stem cells," Nat Biotechnol, published online 30 May 2010, doi:10.1038/nbt.1629.
[6] S. Rodin et al., "Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511," Nat Biotechnol, published online 30 May 2010, doi:10.1038/nbt.1620.
[7] L.A. Couture, "Scalable pluripotent stem cell culture," Nat Biotechnol, 28:562-3, 2010.