Many tools—from transfection devices to off-the-shelf cell lines—push ahead the science of self-renewal.
Despite the political battles, especially in the U.S., over which stem cells can be used in federally funded research, this field continues to expand. In fact, many of this area’s more troublesome steps can now be attacked with stock kits and supplies. Consequently, even more researchers can turn stem cells into scientific results and even applications.
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This cluster of iPS cells, stained for the Oct4 protein, was developed using the CytoTune-iPS Reprogramming Kit. (Source: Life Technologies) |
To turn ordinary cells into stem cells, researchers add genes through transfection, often mediated by a lentivirus or retrovirus. For induced pluripotent stem (iPS) cells, Life Technologies recently unveiled its CytoTune-iPS Reprogramming Kit, which utilizes a Sendai virus. According to David Welch, senior market development manager for the stem cell business unit at Life Technologies, this kit drives cell conversion that is up to 100-fold more efficient than traditional methods and only requires a single application.
Moreover, the Sendai virus does not integrate into the genome of the cells being reprogrammed. “Techniques in which the virus is integrated can cause problems downstream,” says Welch. For example, the integrated DNA might disrupt gene functions in the cell.
Precise Control of the Stem Cell Microenvironment at the Single Cell Level
By Barb McKittrick
The ability to precisely control cellular microenvironment will facilitate a better understanding of cellular behavior and enable many stem cell applications. NanoInk provides a novel tip-based patterning system, NLP 2000 System, which enables the construction of sub-cellular scaled features of multiple materials, allowing the manipulation of the microenvironment at the single cell level. This could be especially important for rare cell types or heterogeneous cell populations.
Microenvironmental control is demonstrated by controlling the shape of cells with patterns of extracellular matrix proteins. Patterns of fibronectin were constructed on glass surfaces with varying geometric shapes and the attached cells were demonstrated to be constrained by pattern geometry (see figure). Precise control of cell cluster size has also been demonstrated by varying the total patterned area. Cluster size is known to play a crucial role in cell function for many cell types.
Finally, preferential binding of cells to different ECM proteins can be utilized to develop single cell co-cultures that facilitate the study of stem or progenitor cell function. Unlike conventional co-culture techniques, the NanoInk platform can spatially manipulate multiple cell types at single cell levels on a substrate, allowing the user to better control co-culture experimental variables. As proof of concept, co-cultures of 3T3 fibroblasts with C2C12 myoblasts were established within microns of each other by binding these cells to fibronectin and laminin features deposited on glass slides at sub-cellular scales. It is conceivable that similar co-culture experiments using stem cells attached to relevant binding domains will contribute to the study of stem cell function and differentiation.
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A New Method for Manipulation
To improve the odds of transfecting stem cells, many researchers rely on electroporation—essentially a shock that creates open pores in the cell membrane. “It is an easy-to-use and reliable method for all cells, on average,” says Michelle Collins, senior global product manager at Bio-Rad.
Collins adds” “Electroporation can be tweaked and optimized for specific cells.” For example, a researcher can adjust the voltage, capacitance, and even the waveform. The combination that works best, though, depends on the cells at hand. “The waveform makes a pretty nice difference in some cells,” Collins says, “and no difference in other cells.”
Bio-Rad Gene Pulser Electroporation Systems give users these adjustments. In addition, these systems can be used with cuvettes or plates, including 12-, 24-, and 96-well plates. It also includes a protocol that works with many stem cells, and can be used as a starting point for fine-tuning.
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The Thermo Scientific Nunclon Vita stem cell culture surface is an energy-treated polystyrene surface designed especially for human embryonic stem cells and iPS cells. (Source: Thermo Fisher Scientific) |
Creating Consistency
When using embryonic stem cells or iPS cells, getting to the desired terminal cell—for instance, a cardiomyocyte—takes time and trial and error. “Cell lines don’t all grow identically,” says Chris Parker, chief commercial officer at Cellular Dynamics International. “So you need to optimize the process on a per-line basis and have different recipes for making particular terminal cells.”
Beyond getting the desired cells, researchers often want lots of them in a pure population. “Many methods to make terminal cells generate a heterogeneous population,” Parker says. “Thus, you may get a mixture where the cell type that you want only makes up 50 to 60 percent of the population, which skews research results. You want to aim for a highly pure population.”
To simplify these challenges, Parker and his colleagues offer various human iPS cell products. For example, they make cardiomyocytes and media for growing them. Parker says that this product is “routinely greater than 98 percent pure cardiomyocytes.” Neurons, endothelial cells, and hepatocytes are in the product pipeline for release later this year.
Building a Complete Platform
Some companies provide a collection of stem-cell tools. For example, Collins says that Bio-Rad covers the complete workflow for stem cells. “We cover cell counting to electroporation to sample preparation and real-time PCR—the entire workflow,” she says.
Thermo Fisher Scientific also provides a range of tools that assist stem-cell scientists. For example, Cindy Neeley, PhD, field technical specialist at Thermo Fisher Scientific, says, “Scientists don’t have a consistent and easy-to-use culture system for the expansion of human pluripotent stem cells.” To help with this, researchers can use the Thermo Scientific Nunclon Vita stem cell culture surface, which is an energy-treated polystyrene surface designed for human embryonic stem cells and iPS cells. This system comes in a 6-well format, and is animal component-free.
Roberta Morris, product market director, life science - cell culture at Thermo Fisher, adds that “customers have used this surface for a number of mouse iPS cell lines, and they have seen enhanced performance for mesenchymal stem cells.”
Beyond working with stem cells, researchers need ways to store them. For that, Thermo Fisher offers several products. For example, the stem cells can be frozen at the desired rate of –1 degree Celsius per minute in the Thermo Scientific Nalgene Mr. Frosty line of freezing containers, which are compatible with all Thermo Scientific Nunc and Nalgene cryogenic storage tubes. Moreover, with the company’s 13×13 cryogenic storage box, coupled with 1 milliliter Cryobank storage tubes, a researcher can double a freezer’s storage capacity, according to Kacey Wiley-Pouliot, the product manager for cryopreservation at Thermo Fisher.
Going 3D
“In the body, cells grow in three dimensions, and they have complex interactions with neighboring cells, which is lost in two-dimensional cell culture,” says Stefan Przyborski, PhD, professor of cell technology at Durham University and director and chief scientific officer at Reinnervate. With Reinnervate’s alvetex, researchers can culture cells in three dimensions. This polystyrene material makes a scaffold in which cells can grow.
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This neural tissue arose from the differentiation of neuroprogenitor cells treated with ec23. (Source: Reinnervate) |
Researchers can purchase multiwell plates that have a disk of alvetex in each well or an insert that fits in a wide range of third-party plates. Reinnervate also makes a holder that suspends the alvetex in a Petri dish, which supplies enough media for long-term, uninterrupted growth.
Researchers can even combine these three-dimensional culturing tools with Reinnervate’s ec23, which is a synthetic retinoic acid. “Natural retinoic acid is often used with neurons, but it’s unstable,” says Przyborski. “So you’re never sure what concentration you have.” Variations in retinoic acid affect neuronal differentiation. With ec23, researchers get the potency of natural retinoic acid, but it’s stable.
Creating New Conditions
As the use of stem cells expands, scientists find more ways to make them, and more kinds of cells to make—some working better in some cases than in others. “One should pick the stem cell type that best helps address the research or clinical challenge at hand,” says Evan Snyder, MD, PhD, professor and director of the Sanford-Burnham Medical Research Institute’s Stem Cell and Regenerative Biology program. Snyder and his colleagues developed Induced Conditional Self-renewing Progenitor (ICSP) cells, which they described in the 4 March 2011, Proceedings of the National Academy of Sciences. Using a viral vector, the scientists inserted just one regulatable self-renewal gene—v-myc—into neural progenitor cells, which could self-renew indefinitely, but only when v-myc was turned on in culture.
The ICSP cells come from cells committed to the nervous system. These neural progenitor cells are induced to self-renew when experimenters turn on the self-renewal gene in vitro by using a particular type of culture medium. When not in that medium, or after being transplanted into the nervous system, the ICSP cells cease dividing and differentiate into particular nervous-system cells.
As today’s stem-cell tools open new avenues of basic research, they also promise therapies for tomorrow.