Researchers face the dual challenges of difficult to handle and hard to maintain stem cells.By Jeffrey M. PerkelTo a certain extent, when it comes to cell culture, one cell is the same as any other. But that's not true of stem cells. Prized for their ability to differentiate into all or just some cell lineages of the body (called pluripotency or multipotency, respectively), stem cells are, as a rule, notoriously finicky. Treat them right, and they will retain their "stem-ness" indefinitely; but let them get too confluent, or change their media conditions, or maybe just stress them out, and they will start differentiating.
 MilliTrace CX Nestin GFP reporter human neural stem cells from Millipore expressing Nestin, GFP and Sox-2. (Source: Millipore)
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For the researchers who study them, stem cells present both challenges and opportunities. On the plus side, stem cells can expose the molecular circuitry underlying cell development programs, provide a platform for drug development and toxicology studies, and potentially serve as therapeutics, for instance, by replacing lost or damaged insulin-producing cells in diabetic patients.
The challenge: human stem cells are considerably more difficult to handle and maintain than standard immortalized cell lines. Researchers have yet to establish consistent guidelines and methods for growing some multi- and pluripotent cells, much less for inducing their differentiation into specific cell types.
"We're sort of in the ‘eye-of-newt' stage," quipped Clive Glover, Senior Product Manager for Pluripotent Stem Cell Biology at
STEMCELL Technologies. "People take eye of newt, add it to cells, and see what happens to make the cells differentiate."
 Differentiated progeny neural stem cells, fixed and stained with the GFAP antibody (an astrocyte marker gene), TRITC phalloidin (actin, red) DAPI (nuclei, blue). (source: sigma-Aldrich Corp.)
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The trick is to maintain the cells in their stem-like state until you want them to differentiate, and then to direct that differentiation process to yield a desired cell population—for instance, a particular kind of neuron or islet cell.
Despite these challenges, stem cell research, on both embryonic and adult stem cells, is progressing at a blistering pace—perhaps no more so than with so-called "induced pluripotent stem cells" (iPS cells). First described in 2006 by Shinya Yamanaka of Kyoto University, iPS cells are differentiated cells (such as skin cells) that have been essentially reset into something akin to an embryonic stem cell (ESC), via the introduction of four protein-coding genes. Because they involve neither the creation nor destruction of humans embryos, these cells neatly sidestep the ethical concerns typically associated with ESC research, and researchers have embraced iPS cells wholeheartedly. According to the ISI Web of Science, Yamanaka's original iPS publication has been cited more than 800 times in just three years.
Stem cell tool manufacturers have responded with a range of media and reagents for the culture, maintenance, differentiation, and analysis of adult and embryonic stem cells.
Cell MaintenanceOne of the primary concerns for researchers pursuing stem cell research, especially with ESCs, is that most growth media formulations are neither fully chemically defined nor free of animal components. Traditionally, ESCs are cultured on mouse embryonic fibroblast (MEF) feeder cells, and in the presence of sera. But cell lines and sera vary from batch to batch, limiting reproducibility and making eventual regulatory clearance problematic. Further, MEFs must be cultured and maintained separately, piling on additional time in the culture hood. In any event, animal components are undesirable if stem cells are ever to progress to the clinic, as they pose the risk of inadvertent animal pathogens.
Considerable effort has, therefore, been spent developing culture conditions and reagents for feeder-free and serum-free stem cell work. One solution is to use media that has been conditioned by growth of mouse or human fibroblasts. Alternatively, researchers can use growth layer scaffolds, such as
Life Technologies' CELLstart matrix or
BD Biosciences' BD Matrigel.
A tumor-derived extracellular matrix extract that provides a scaffold for cell growth on the culture dish, BD Matrigel is popular among stem cell biologists. But BD actually offers several feeder cell replacements, including BD Laminin/Entactin. "The advantage is that it is purified," says Charles Crespi, vice president Discovery Biologicals at BD Biosciences. "It isn't fully chemically defined, but it is a highly purified product" that provides a consistent surface for cell attachment. Or, researchers can try BD PuraMatrix, a growth factor-free hydrogel, which Crespi describes as "like a blank canvas" for 3D cell culture; growth modulators may then be added back in defined ways.
 Nanog staining on human ES cell line, BG01V, grown on irradiated mouse embryonic fibroblasts. Nanog is in green and it is co-stained with DAPI in blue. This staining uses a Nanog antibody that is directly conjugated to a fluorescent dye. The catalog number is NL1997G. (Source: R&D Systems)
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Companies provide specialized growth media, too.
Sigma Aldrich's Stemline culture media provides serum-free culture conditions for adult stem cells, while Life Technologies offers a partially defined serum product called KnockOut SR serum replacement (in both animal product-containing and "XenoFree" formulations). Life Technologies' STEMPRO hESC SFM, and STEMCELL Technologies' mTeSR1 and animal protein-free mTeSR2 are fully defined for use in both serum- and feeder-free conditions;
Millipore's HEScGRO Media is also fully defined, but relies on the use of human feeders to provide a serum- and animal-free environment.
Cell DifferentiationIn general, the process of coaxing stem cell differentiation is a simple matter of switching media. By adding and/or removing growth factors, you can induce the cells to begin a process of terminal differentiation. The trick is to guide that process to make the cell type you want. STEMCELL Technologies' AggreWell helps standardize ESC differentiation, by simplifying the process of making so-called embryoid bodies (EBs). EBs are "3-D balls" of ESCs, which subsequently differentiate. They are made by scraping cells off a plate; as a result, they are highly variable, making downstream analyses difficult. "You typically get a very heterogeneous mix of sizes," says Glover. AggreWell uses fixed-sized microwells in a 24-well format to create relatively uniform EBs. As a result, says Glover, "you can standardize the way you make EBs, and end up with a much more homogeneous population."
R&D Systems and Millipore both offer kits for differentiating neural and mesenchymal stem cells into oligodendrocytes, astrocytes, and neurons, and chondrocytes, osteocytes, and adipocytes, respectively. R&D also offers a system for differentiating human ESCs specifically into dopaminergic neurons.
Monitoring PluripotencyWhether your goal is to maintain stem cell pluripotency or induce differentiation, you need a way to monitor the cell population—that is, to do some culture quality control—and several classes of product are available to do that.
Beckman Coulter offers multiplex RT-PCR panels of 24 to 30 pluripotency markers that can be used with the company's GenomeLab GeXP Genetic Analysis System to assess iPS cell reprogramming.
"When somatic cells are reprogrammed, individual ESC-like colonies must be characterized to determine their pluripotent potential. This requires time and the cost of reagents to expand and assay them," says Kathryn Sciabica, Senior Applications Scientist for Genetic Analysis at Beckman Coulter. "With our system, you can get a lot of information from very few cells, saving both time and money."
 H9 cells preserve pluripotency post Nucleofection. H9 cells transfected with the pmaxGFP Vector maintain their undifferentiated state. Analysis after 24 hours shows expression of GFP (green) as well as of the pluripotency markers SSEA4 (red) and Oct4 (purple). The blue signals refer to nuclear staining by DAPI. (Source: Jennifer Moore, Rutgers University)
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R&D Systems provides similar information via a filter-based antibody array that can test cell lysates for markers of pluripotency, plus each of the three differentiated germ layers (endoderm, mesoderm, and ectoderm), "so you can make sure the cells remain pluripotent," says Joy Aho, a scientist in the stem cell department at R&D Systems.
Playing on its strength in flow cytometry, BD Biosciences offers antibody panels for flow-based analysis and sorting of stem cell populations based on, for instance, Oct3/4, SSEA-1, and SSEA-4. Flow cytometry, says Robert Balderas, BD Biosciences' vice president of Biological Sciences, provides two important benefits over alternative methods of monitoring differentiation. First, users can actually count how many cells are either pluripotent or differentiated (RT-PCR, in contrast, analyzes the bulk population of cells in aggregate.) Second, users can, if desired (and equipped with a cell sorter and antibodies specific for cell surface receptors), "subdivide a heterogeneous population to sort out, with very high purity, the populations of interest."
Functional AnalysesCompanies provide a variety of genetic tools and reagents for functional manipulation and analysis of stem cells. Sigma Aldrich and
Thermo Fisher Scientific, for instance, offer libraries of short-interfering RNAs (siRNA) and short-hairpin RNAs (shRNA) for probing gene function via RNA interference (RNAi). Alternatively, users can try targeted gene "editing," using Sigma Aldrich's CompoZr line. Based on technology developed at
Sangamo Biosciences, CompoZr zinc finger nucleases (ZFNs) use specially designed chimeric proteins to catalyze genetic knock-out or knock-in reactions
in vivo.
To deliver nucleic acids, users can opt for any of a variety of gene delivery approaches, from lipid-based transfection, to viral transduction, to electroporation.
Lonza's amaxa nucleofector system and human stem cell "starter kit" can transfect human ESCs with "high transfection efficiencies combined with excellent viability and functionality of the transfected cells," says Gerhard Muster, Product Manager at Lonza Cologne AG, and are suited to transfect iPS cells as well. However, adherent cells like hESCs must still be detached before transfection; Lonza will be rolling out a product supporting adherent cell transfection to overcome any potential problem caused by this step.
For those interested in human neuronal stem cells, Millipore offers GFP-tagged ReNcell reporter cell lines in which GFP expression is either constitutive or differentiation-dependent; in the latter case, GFP is controlled by the nestin promoter, which is active in pluripotent neuronal stem cells but turns off during differentiation.
"When the cells are in the stem state, they are green," explains Vi Chu, Manager of R&D for the stem cell/cell biology team at Millipore; "as we switch to differentiation media, the GFP starts to downregulate and damps its fluorescence."
According to Chu, researchers can use these cells to screen small molecules to determine whether they affect stem cell maintenance or differentiation. "More and more researchers are using these as models for screening or toxicology studies," she says.
Despite the pace of stem cell research and the plethora of tools available to those who are conducting it, several unmet needs remain. "There is this overwhelming need to come up with uniform methodologies to differentiate and grow the cells," says Chu. That's certainly true of iPS cell development, says Carl Schrott, marketing director in the Regenerative Medicine group at Sigma Aldrich. "There is a lot of interest in iPS cells, but there are only a few labs that know how to make them," he says.
Animal product-free culture, especially, remains an area of intense research, adds Crespi. "Nobody has a completely animal-free solution from soup-to-nuts, it's very early days pretty much everywhere."
Also needed, says Balderas, is a better understanding of the cell surface markers that define various differentiation states, so pluripotent cells may be distinguished (and separated) from more-differentiated cells.
"I would say the entire community of stem cell biologists is in a very special space," Balderas concludes. "It moves very fast, faster than anything we have ever seen."