by Jeffrey M. Perkel
It’s an exciting time for human embryonic stem cell (hESC) researchers.
In January, the US Food and Drug Administration gave Geron Corp. the green light to begin the world’s first clinical trial based on these controversial biologics. Work on induced pluripotent stem (iPS) cells, less-contentious alternatives made by reprogramming adult somatic cells into something akin to hESCs, continues to advance in sophistication and safety. And earlier this month, to the delight of the scientific community, US President Barack Obama rolled back restrictions on federal funding of research using hESC lines derived after August 9, 2001.
Unfortunately, hESC research isn’t easy—in fact, it is anything but; hESCs are the double black diamond runs of cell biology.
“Technically, hESC are much more challenging to grow than [for instance] HeLa cells,” says Karl Willert, director of the hESC core facility at the University of California, San Diego.
But help is coming. There now exists a range of tools and techniques for the maintenance, characterization, and differentiation of these cells.
Perhaps the most critical, but also basic, tool for hESC work is the media in which the cells are grown. hESCs aren’t your garden-variety immortalized cells; they are pluripotent, and must be maintained in an undifferentiated state. Yet precisely because they are pluripotent—that is, they can differentiate into any of the three primary germ layers: endoderm, ectoderm, and mesoderm—maintaining hESCs requires tremendous commitment.
“hES cells require a lot of care and sensitive handling,” says Vi Chu, R&D manager for the stem cell team at Millipore.
Whereas many traditional cell lines are perfectly content to be grown to confluence or left unattended for days, hESCs are downright persnickety. Let them get too dense, or change their growth conditions even slightly, and they can begin to differentiate. As a result, they require daily care and feeding.
Initially, hESCs were grown on a layer of feeder cells (usually mouse embryonic fibroblasts, or MEFs) in growth media supplemented with fetal bovine serum and growth factors. These conditions present two basic problems. First, between the serum and the MEFs, the growth media basically constitutes a black box; day-to-day and lot-to-lot variability is inevitable. Second, the use of ill-defined and animal-derived components could present future regulatory problems for those who might wish to move their research into clinical development. As a result, there is considerable effort towards the development of fully defined, animal product-free media.
One popular choice, says Willert, is Invitrogen’s KnockOut™ SR serum replacement, a partially defined, animal product-containing derivative of fetal bovine serum; a XenoFree variant, the KnockOut™ SR XenoFree serum replacement, was released in December, enabling researchers to culture hESCs in the absence of non-human components.
Other hESC-specific media include Millipore’s HEScGRO, Invitrogen’s STEMPRO hESC SFM (serum- and feeder-free medium), GlobalStem’s Ready-for-Action ES-DMEM/F12, and STEMCELL Technologies’ mTeSR™1. Many are fully defined and require no serum, and some enable feeder-free growth as well. mTeSR™1, at least, supports iPS growth; in fact, according to Clive Glover, senior product manager for pluripotent stem cell biology at STEMCELL Technologies, the iPS cell bank at WiCell “entirely uses mTeSR™1 to maintain their bank.”
Willert’s facility frequently opts to supplement its media with human serum albumin, a clinical product from Bayer, in addition to Knockout SR and basic fibroblast growth factor (bFGF) from Life Technologies.
“The cost of FGF is one of the major costs of working with these cells,” says Willert. “You have to feed the cells every day, and add fresh FGF each day.”
Feeder cells play a dual role in hESC culture. They provide both a growth substrate and growth factors. The latter can be swapped out for exogenous cytokines. To replace the former, some researchers use basement membrane replacements, such as BD’s Matrigel or Invitrogen’s Geltrex. Invitrogen also offers a xeno-free option, the CELLstart™ substrate.
Those researchers who choose to use MEFs can either derive their own, again leading to potential batch-to-batch variation, or buy them from such companies as GlobalStem and Millipore. For GlobalStem, commercial MEFs inject some much-needed standardization to the field.
“The field has been developing quickly and is relatively young, so standards have been lacking,” says Founder and President Jonathan Auerbach.
According to Amy Laws, associate product manager at BD biosciences discovery labware, whether a researcher opts to use feeders or not, “all surfaces need to be qualified for their ability to maintain pluripotent hESCs.” BD, in collaboration with STEMCELL Technologies, offers “a pre-qualified complete environment” for that purpose, comprising BD hESC-qualified Matrigel Matrix and mTeSR™1 media.
Several companies offer reagents to assess the pluripotency of growing hESC cultures, especially antibodies for such intracellular and cell surface hESC markers as the SSEA and TRA proteins, Oct4, and nanog.
BD Biosciences, for example, offers a kit containing fluorescently labeled antibodies against TRA-1-81, SSEA-1, and SSEA-3. “The first two are markers of pluripotency, the last is a marker of differentiation,” explains Robert Balderas, vice president of biological sciences at BD. Using this kit, researchers can both assess the degree of pluripotency in their hESC cultures, and—with the help of a cell sorter—enrich for pluripotent (or differentiated) cells. Millipore’s FlowCellect Human ESC Surface Marker Characterization Kit performs a similar analysis based on expression of HESCA1, SSEA1, and SSEA4.
Alternatively, researchers can assess “stem-ness” via RT-PCR, for instance using Invitrogen’s StemPro EZChek, a multiplex assay that measures the expression of Oct4 (a marker of pluripotency), AFP, ACTC1, and Sox1 (markers of endoderm, mesoderm, and ectoderm, respectively), and GAPDH (an internal standard).
Those who wish to differentiate hESCs can use STEMCELL Technologies’ AggreWell™ plates. AggreWell™ attempts to normalize one method of hESC differentiation, in which cell clumps are manually scraped from a culture dish and grown in suspension. The problem with this approach, says Glover, is that this method is highly heterogeneous and irreproducible. AggreWell™ “is an attempt to bring standardization to the process of making embryoid bodies,” he says.
The product is basically a modified 24-well culture dish in which the wells contain 1,200 tiny inverted pyramids—microwells—that can capture individual cells and grow them into individual embryoid bodies. The process, Glover explains, is no faster than the traditional method, “but you gain so much in terms of control.”
Thermo Fisher Scientific caters to those interested in functional analysis of stem cell biology. The company, via its Dharmacon and Open Biosystems product lines, offers libraries of short interfering RNAs, short hairpin RNAs, cDNAs, ORFs, microRNA inhibitors, and microRNA mimics, to either raise or lower the expression of virtually any gene or regulatory RNA in the cell. (The company also provides methods for detecting differentiation markers via antibodies and PCR through its Pierce and ABgene product lines.)
Devin Leake, director of R&D for Thermo Fisher Scientific’s genomics group, explains the utility of such tools by citing an internal study using mesenchymal stem cells (MSCs).
“Using miRNA inhibitors or mimics, we identified three microRNAs that induce the MSCs to differentiate into osteocytes,” Leake says. “Controlling stem cell differentiation through the use of microRNA, allows researchers to study the process of differentiation as well as osteocyte biology.”
“Using genomics tools enables researchers to regulate the biology,” he concludes.
To deliver nucleic acids to stem cells, researchers have several options. They can use lentiviral vectors (kits are available from both Thermo and Invitrogen), chemical transfection agents (such as Life Technologies’ Lipofectamine or Thermo’s DharmaFECT reagents), or electroporation (for instance, using BioRad’s electroporator or amaxa’s Nucleofector). Thermo also offers a line of chemically modified siRNAs called Accell siRNAs that can be taken up by cells without a transfection reagent.
Finally, to freeze down hESCs, researchers have several dedicated options, including GlobalStem’s hESfreeze and STEMCELL Technologies’ mFreSR™ cryopreservation media. As with mTeSR™1, mFreSR™ is fully defined and serum-free, which is a plus for those who also maintain their cells in the absence of sera, says Glover. “If you’re trying to eliminate undefined components, cryopreserving in serum is not optimal.”
Considering the excitement surrounding them, hESC researchers have a surprising number of critical unmet needs, including standard culture conditions, protocols for differentiation into desired cell types, and reporter cell lines that, for instance, activate GFP when they reach some developmental milestone.
“It’s a young field,” says Willert, “we have to come up with protocols that work for everyone and optimize these protocols on human embryonic stem cell lines.”
Still, at the pace the research, and the tool development, is progressing, it likely won’t be long before at least some of those gaps are filled.