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High-Throughput Bio Meets Stem Cells

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Stem cells have captured the attention of the mainstream public, but until very recently they were always the purview of some other biotech discipline. From cloning to potential use as a therapeutic, stem cells have managed to stay just at the periphery of high-throughput biology.

But now, genomic technologies are taking stem cell research to even more promising levels. For the first time, thanks to these tools, scientists have been able to truly analyze the induced stem cells (consider these the we-really-hope-this-is-just-like-a-stem-cell cells) that researchers created from adult skin cells. In this and many other ways, genomics and proteomics are proving that they too have something to offer in the study of these fascinating cells.

So far, epigenomics has been the most useful in these pursuits. Analyses of methylation patterns and histone modifications have been instrumental in determining the pluripotency state of stem cells, and chromatin immunoprecipation work — both with microarrays and with sequencing — has gone a long way to revealing the transcription factors in play. Meantime, microRNA research has begun to take hold as scientists evaluate how these small RNAs help control the stem cell differentiation process.

Wishful stem cells

The ethical and political landmines surrounding human embryonic stem cell research — not to mention the practical issue that there simply aren't all that many of these cells to study — has long left researchers looking for a better way to generate as many of these vital cells as possible.

Enter Kyoto University's Shinya Yamanaka, who in 2006 published breakthrough work demonstrating that four transcription factors could be transfected into mouse skin cells to turn them back into cells that very closely resembled embryonic stem cells. He called them iPS, or induced pluripotent stem cells. He and another team, led by James Thomson at the University of Wisconsin, followed that last year by duplicating the feat using human fibroblasts. Not only do iPS cells promise more yield when it comes to using them for regenerative medicine, they also offer a convenient alternative to researchers hoping to sidestep legislation that prohibits the use of cells derived from live human embryos and cloned cells.

The initial study, however, had its limitations — the biggest being that it didn't prove that the cells were functionally the same as embryonic stem cells in being truly pluripotent. Last year, among several follow-up studies, a collaboration between Harvard's Konrad Hochedlinger and Kathrin Plath at the University of California, Los Angeles, showed that by using a revised protocol and large-scale biological assays, they could derive pluripotent versions of these cells that were functionally identical to human embryonic stem cells.

Most of the work characterizing stem cells has revolved around epigenetic and epigenomic changes, since in the end, these are the factors that control cell fate within an organism. "Our main focus was studying the epigenome of the cells … because we knew that from previous experiments in using nuclear transfer, [or] cloning, that epigenetic abnormalities are the principal reason for the failure of cloned animals," says Hochedlinger, who splits his time between Massachusetts General Hospital and the Harvard Stem Cell Institute in Boston. "By improving the technology, we were able to generate cells that were epigenetically, and also functionally, indistinguishable from embryonic stem cells."

His first step was to select cells that had been successfully reprogrammed. He identified two genes necessary for a cell's survival, Oct4 and Nanog. By running the cells through an assay to see if they lived, he could determine whether Oct4 and Nanog were active, a signal of faithfully reprogrammed iPS cells. "These markers are essential for embryonic stem cells — if you delete the genes the embryonic stem cells die," Hochedlinger says. "The difference [in] our approach was that the marker we've used is a critical gene for embryonic cells and using that one … yielded cells that are indistinguishable from embryonic stem cells."

With these iPS cells in hand, Hochedlinger then moved to focusing on the epigenome at three separate levels: gene, chromatin, and full genome. In the first assay, he used bisulfite sequencing to determine whether the promoter regions of Oct4 and Nanog were methylated. Normally, says Hochedlinger, these promoters are "heavily methylated in skin cells, which correlates with their repression, but then demethylated in embryonic cells, which correlates with their re-activation. We found in our iPS cells these promoter regions are demethylated and the genes are expressed."

To probe chromatin reprogramming, the team utilized the property of X chromosome inactivation. During development in mammalian females, one of the two active X chromosomes is deactivated. Hochedlinger used a female skin cell line where one of the chromosomes was tagged with a green fluorescent protein marker. When he introduced the four transcription factors into cells where the deactivated X chromosome had been tagged, the formerly silenced X chromosome lit up. "We concluded that epigenetic reprogramming had occurred not only at the gene-specific but also the chromosome-wide level," he says.

Finally, he used a ChIP-chip assay to look at histone modifications on a genome-wide scale, focusing on two key ones: histone H3 lysine 4 and histone H3 lysine 27, shown to be correlated with active and repressed genes, respectively. The researchers found that the patterns between reprogrammed cells and embryonic stem cells were, for all intents and purposes, identical, says Hochedlinger. While bisulfite sequencing and X chromosome inactivation are not new, the more recent ChIP-chip technology enabled Hochedlinger and Plath's group "to go one step further to ask whether reprogramming had really occurred at the genome-wide level," he says.

Tracking ChIP

The availability of high-throughput chromatin immunoprecipitation technologies has paved the way for a number of advances in stem cell research. One early adopter to apply ChIP-seq to his large-scale chromatin mapping studies is Mass Gen's Brad Bernstein, whose work on bivalent chromatin in stem cells was a turning point for the field.

Unlike in DNA methylation, which is associated with gene silencing, histone modifications occur on specific amino acid residues of chromatin. In some cases, histone methylation leads to expression, and in other cases, to gene silencing. "If you methylate lysine 4, this typically is associated with activity; if you methylate lysine 27, this typically is associated with repression," Bernstein says. "Now the combined pattern of lysine 4 and lysine 27 is associated with repression, but we think it is a special state — it's a less stable, it's a plastic state. It's poised for coming on later."

In previous work, Bernstein used ChIP-chip analysis to construct a genome-wide map of chromatin state in stem cells, and found that certain highly conserved, developmental regulator loci were associated with bivalent chromatin. This gave him the idea that bivalent chromatin marks keep differentiation genes off in embryonic stem cells in order "to keep the embryonic stem cells poised for alternate fates," he says. "By tracking how those promoters flip and switch between states as cells differentiate, one can learn a lot about the state of the cell and about the potential of the cell."

In a recent study, Bernstein and his team again looked at these modifications, this time using a ChIP-seq assay. The two main benefits of  using the ChIP approach with next-gen sequencing, Bernstein says, are that it's less expensive and it requires less DNA. "High-throughput sequencing … is inherently genome-wide, and you only need a few nanograms of DNA for the analysis," he says.

Major microRNAs

Transcription factors, DNA methylation, and histone modifications aren't the only elements that control gene expression in stem cells; microRNAs are another player. One of the larger challenges to studying stem cells is the difficulty in identifying separate lineages in embryonic stem cell lines and varying temporal stages of differentiation among tissue stem cells. This is where miRNA transcription profiling has gained solid footing — and possibly made strides ahead of the traditionally studied epigenetic factors, since miRNA expression is more closely correlated with protein translation.

UC San Diego's Louise Laurent, for example, has been using miRNA arrays for genome-wide miRNA profiling studies. In a recent collaboration with Jeanne Loring, founding director of the newly created Center for Regenerative Medicine at The Scripps Research Institute, Laurent performed miRNA profiling on a collection of several cell lines, including human embryonic stem cells, fetal neural stem cells, and adult neural stem cells. "We definitely found a unique profile in the human embryonic stem cell [line], so we could easily distinguish them from all the other cell types," Laurent says. Collaborators also found that the adult and fetal neural stem cells "were very easily distinguishable by microRNA profiling, something that based on normal phenotypic characteristics is difficult to do." In the human embryonic stem cell profile, they found that many of the same miRNAs that are up-regulated in these cells have the same seed sequence, "suggesting that there's kind of a ganging up of multiple microRNAs in embryonic stem cells, potentially — and this is the part that we're working on now — to regulate a very important network of genes."

Laurent used an miRNA microarray "because it gives very good and robust measurements in terms of relative amounts," she says. "But it doesn't really measure absolute amounts reliably." For validation, her team typically uses RT-PCR and is starting to transition to sequencing. The drawbacks, of course, are that "it's relatively new, it's relatively expensive, and so at this point it's difficult to do an experiment on the scale that ours describes with sequencing," she says.

Laurent focuses primarily on human embryonic stem cells, but most of the current work in miRNA transcription profiling has been done in mouse embryonic stem cells. They're easier to manipulate than human cells, she says, and "at the end of the day, it's actually a bit difficult to test what they're capable of. The gold standard in the mouse realm is to make a mouse out of it, and we obviously can't make a human."

Being armed with expression data is helpful, but finding functional hits — that is, which mRNAs are targeted and how downstream protein levels are affected — is really the first step toward applying any of this basic research in the clinic. At UC San Francisco's Gladstone Institute of Cardiovascular Disease, Deepak Srivastava has combined miRNA microarray expression profiling and RNAi knock-in techniques using shRNA lentiviral vectors to study the effects of two muscle-specific miRNAs, miRNA-1 and miRNA-133, on differentiation of mouse embryonic stem cells. He recently found that both miRNAs actively push the stem cells toward mesoderm fate and also repressed non-muscle cell fate gene expression during differentiation.

"The miRNAs are pushing cells to become mesoderm and then muscle, but at the same time they're telling the cell to not turn on genes that would normally be present in endoderm and ectoderm," Srivastava says. "It's shutting off genes and keeping these cells from going into other cell types." Being able to control the differentiation process and either direct it or keep cells from differentiating is key to creating stable banks of desired cell types. And while the field is still new to discovering translational control by miRNAs, Srivastava thinks it's only a matter of time before the true scope of their activity is found. "I think it's likely that the same paradigm will hold true for not just muscle but many different cell types; their fate will be regulated by one or more miRNAs."

Not only are miRNAs working downstream to turn on or off gene expression in differentiating cells, they're also being turned on or off by upstream factors. In a collaboration with George Daley, Richard Gregory's lab at the Harvard Stem Cell Institute found that a conserved RNA-binding protein called Lin-28 binds to the let-7 family of miRNAs, keeping them silenced in embryonic stem cells and thereby blocking differentiation. In fact, Lin-28 is one of the four so-called "pluripotency factors" that James Thomson used in his follow-up study to Yamanaka's work, transforming human skin cells back into iPS cells.

"It seems that perhaps inhibiting let-7 in the skin cells by introduction of Lin-28 facilitates this de-differentiation back to the pluripotent state," Gregory says. "These different pathways are all kind of converging now — miRNAs, dedifferentiation, stemness, and all of those things. In part, this is all mediated through Lin-28."

Gregory focused on Lin-28 because he found that it was highly expressed in stem cells, but not in other cell types. After isolating Lin-28, his team ran a combination of both in vitro and in vivo assays, including RNAi and miRNA microarrays, to show that this factor specifically blocked only members of the let-7 family of miRNAs.

In part, follow-up studies will focus on finding the target genes of let-7, a process that represents one of the bigger problems, Gregory says. He sees future functional work moving toward mass spectrometry approaches to measure changes in protein levels in response to different miRNAs. "It's a bit tricky with the current technologies to actually identify an miRNA target gene," he says. However, "you might actually miss some targets simply by looking at the RNA level. Looking at the protein level may be a better readout."

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Where It's All Going: The Clinic

The end goal of all this research, whether in adult or embryonic stem cells, is creating effective therapies. The field of regenerative medicine has seen a huge boost with the advances in both culturing human embryonic stem cells as well as converting somatic cells into iPS cells. The larger challenge for this field, however, remains: acquiring a large enough number of cells that also won't be rejected upon transplant into a human body.

It's not only embryonic stem cells that are difficult to procure. Margaret Goodell, director of the Stem Cells and Regenerative Medicine Center at Baylor College of Medicine, studies hematopoietic stem cells in mice. The difference, she says, is that one can grow embryonic stem cells in culture, but not so for adult stem cells like hematopoietic stem cells. These cells are rare in bone  marrow; so-called cancer stem cells are also few and far between. For the most part she's confined to purifying these bone marrow cells from freshly sacrificed mice since "for [these] cells, there is no good way to simply grow them up in vitro, and there's no good cell line model for hematopoietic stem cells. So we are absolutely limited."

While for her current studies, which involve gene expression profiling, the numbers she extracts from her lab mice are sufficient (50,000 to 100,000 cells from 10 to 20 mice), she would need many more to do any type of proteomics work. For most other types of adult stem cells, such as skin or muscle, she adds, "none of these can you really grow so that you could get huge amounts to do other kinds of studies. So stem cell numbers are certainly going to be a limitation for a while."

Melbourne-based Stem Cell Sciences is focusing on solving this problem with its culture services. With partner research labs located all over the world, its primary service is providing academic and pharmaceutical clients with cultured, terminally differentiated human embryonic stem cell lineages, ranging from neurons to pancreatic islet cells.

"It's primarily for the drug discovery pipeline," says Rob Burgess, VP of business development. One of its corporate partners, Merck, is using the technology and stem cell lines for its own drug research efforts. "Merck is utilizing our neural stem cell platform technology and that is animal-based — and it is utilized primarily for looking at potential drug candidates in this area and how they interact with neural stem cells and the differentiated progeny that result from those," Burgess says.

At the basic research level, being able to characterize stem cells, especially those being cultured for regenerative medicine, is at the heart of all this large-scale genomics work. Whether it's to understand how stem cells grow and differentiate, screen the epigenome of possible therapeutic lines, or determine the exact stage of differentiation — all these are important to pick the best possible cells for patients. The Gladstone Institute's Deepak Srivastava, a cardiologist by training, sees being able to pinpoint cellular state by knowing which miRNAs are turning differentiation on or off as one of the most important challenges for stem cell research. "That's … really important when you think about cell-based therapies," he says. "If you introduce cells, let's say into the heart for treating heart failure, it's really important that those cells only become heart cells and don't become bone or teeth or hair or some other cell type. The ability to not only tell a cell what to be, but tell it what not to be, is really important."

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In the US, Trying to Get Past Politics

In 2001, US President George W. Bush signed into law a moratorium on federal funding for research using human embryonic stem cells, thereby limiting the number of available cell lines. Many labs have studies using both NIH-approved cell lines and other cell lines, but the challenges they face aren't simple. Issues surrounding transparency in how and what they use the money for, as well as infrastructure and practical space constraints, are some of the biggest challenges that US-based stem cell researchers face.

At the Gladstone Institute, Deepak Srivastava works with both mouse and human embryonic stem cells, and in part he's limited by the availability of stem cell lines. For their recent study, he says, "we did use NIH-approved stem cell lines. There are others that are not approved by NIH that are probably better to use, have better growth characteristics, and those fall outside things NIH funding would allow."

A more practical concern is one of logistics. Because most stem cell researchers receive multiple sources of funding, they are studying both NIH-approved and non-approved stem cell lines, for which they have to tailor their lab setups accordingly. "We have to separate the space and equipment to do work on non-NIH-approved cell lines," Srivastava says. "It's a shame that we have to do that."

The state of California found a way around the federal restrictions by passing Proposition 71 in 2004, legislation that has allowed the state to set aside $3 billion in state funds for stem cell research. That's been a windfall for scientists and a boon to the state, which continues to lure researchers with its more open laws.

At the Burnham Institute for Medical Research, that proposition saved a budding stem cell research program that had been hastily rolled out into a wholly owned subsidiary to get around concerns about what federal funding would and wouldn't allow. Evan Snyder, director of the Stem Cells and Regenerative Medicine program, says the Burnham recruited him from Harvard to negotiate the battleground. With California's safe haven law for stem cell research, Snyder says, "They also considered it to be as if the state has thrown down the gauntlet and said, 'OK, we're protecting you, now go for it and build up stem cell programs.' And the Burnham really was one of the first to seize this opportunity and say, 'We really want to build up a Manhattan Project-style stem cell effort.'"

Today, the Burnham and other San Diego-area institutions are working to protect against another funding fiasco. A proposal currently at CIRM, the agency in charge of      doling out the state's $3 billion, would establish an institute jointly owned by Scripps, Burnham, UC San Diego, and the Salk Institute. The idea, says Burnham President John Reed, is to set up a facility without a single dollar of federal money — a research venture that would never be subject to the whims of politicians, and where scientists could pursue what's best for stem cell biology, rather than what's allowed by the funding agencies.