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Model citizens
August 2013
by Randall Willis  |  Email the author


Ralph sits anxiously in an examination room. A few months ago, he was diagnosed with rheumatoid arthritis, just the latest in a string of conditions he gets to worry about. His doctor did a blood draw and sent the samples off for testing. Today, he learns the results.
"Good news, Ralph. We seem to have identified a drug that should help with your RA without interfering with your insulin regimen or increasing your risk for heart disease. "Now, we can't guarantee no side effects," the doctor warns, "but your stem cell tests suggest this is your best option."
Ralph is relieved.  
Although this story is fiction, recent efforts in stem cell research suggest the day may not be too far down the road when a patient's own cells can be used to screen for drugs that work best for his or her condition in the context of comorbidities and concomitant medications.  
Open doors  
When stem cell work was once something of a niche, offering incredible therapeutic potential under an ethical cloud, the advent of induced pluripotent stem cells (iPSCs) and discovery of a variety of stem cell types from adult tissues has blown the doors off this field and generated buzz in numerous research areas. But buzz does not expertise make, and many researchers are finding their ideas overstep their abilities, opening the door for companies like Life Technologies Inc. (LTI).
"This is a very new field, and we thought people probably aren't going to get a lot of expertise, resources or time, so this might also be an area they would outsource," says Carolyn Pettersson, senior manager of LTI Discovery Services.  
To address those needs, the company recently initiated a stem cell service called CellModel.  
"We tie a lot of it back to drug discovery, because we believe that people will want to create different models and cell types and do assay development and screening, and that has been our focus and our strength in services," Pettersson says.  
"One aspect of someone coming to us could be to give us patient samples, which we could sequence using our reagents," adds LTI Associate Director David Piper. "By using these cells, you can get a better understanding of genetic variability as well as the disease state that the samples may come from. We can then take those samples and reprogram and differentiate them to specific lineages, and again, using a multitude of fluorescent detection systems that we have from our Molecular Probes site, build assays and return the data. For a small company to do that is a big licensing commitment, a big infrastructure commitment to start."
As Pettersson further explains, because CellModel is developed using LTI's own products and instruments, the client can be trained on the protocols and take over the process at any point, knowing it has complete access to all of the equipment and supplies they need—a one-stop shop solution, as she describes it.
For Chris Parker, vice president and chief commercial office of Cellular Dynamics International (CDI), which offers an array of cell types through its iCell portfolio, it's a question of build or buy.  
"Do you try to make your own picks and shovels, or do you buy a pick and shovel and start mining for gold?" he asks. "We've found that our customers are better suited and demonstrate more value by finding ways to use these resources as opposed to finding ways to make them."  
Quality was a key component of the equation for Parker.  
"CDI put in a quality system that really managed the materials such that we could truly manufacture these things consistently," he explains. "So, if I was a researcher using a cardiomyocyte model, and you were a researcher using cardiomyocytes in New York, with cells from CDI, we could compare our results. Otherwise, if you made your cardiomyocytes and I made my cardiomyocytes, it's highly likely that they're not going to be the same."  
This is going to be critical for using this data in a regulated environment, he adds, as organizations like the U.S. Food and Drug Administration (FDA) will want to know whether the results are true across the board.  
An added feature, from Piper's perspective, is that stem cells offer researchers the best of all worlds because they offer the opportunity to perform experiments with the ultimate control, the patient.  
Traditionally, experimental controls have consisted of a large diversity of individuals who do not have the disease of interest, and comparing them to the people with disease, he explains. "In the best-case scenario, perhaps those people are related. But another fundamental way of approaching that is to take a disease sample—say you have a monogenic disease—and use a technology like TAL editing or something along those lines to address a single nucleotide in the genome. You change it to make the cell wild-type, and now you have a wild-type control that is genotypically the same except for that single monogenic disease."
It is tantamount to using an identical but healthy twin to understand what has gone wrong in the unhealthy twin and upon whom to test treatments.  
CDI's Parker concurs.  
"We can use that same genetic engineering methodology not only to correct and create what we call isogenic controls, but we can also modify the genome," he suggests. "We can do over- expression to create a disease model within a normal patient and compare it to a disease model from an iPSC patient."  
Among the molecular tools CDI has licensed are LTI's GeneArt Precision TALs (TALENs) and Sigma's CompoZr ZFN technologies, in deals announced in June. Of course, reprogramming an adult cell into an iPSC is not always straightforward.
Integrating non-integration
One of the challenges of developing stem cell lines is not changing the genetic background of the cell while returning it to a pluripotent state, a problem exacerbated by integrative vectors such as most viruses.  
"You want to avoid viruses in general, which are not generally looked at highly by the FDA," says Brad Hamilton, director of R&D at Stemgent. "For reprogramming, there are some DNA vehicles—plasmids, episomes, minicircles or things like that—which you could call a transient state, but they still have the potential to integrate."  
And integration can lead to a series of downstream problems, such as increasing the teratogenic potential of the cells, limiting their future use as either a research tool or therapeutic (more on that next month).  
"We'd obviously seen publications for Sendai and episomes and for other DNA systems, but it wasn't quite what we felt was going to be the ultimate of where we wanted to take things," Hamilton adds. "And dogmatically, we knew mRNA was a way to manipulate cells."  
Stemgent chose to work with Harvard University's Derek Rossi and Luigi Warren, leaders in the development of mRNA for cellular reprogramming, to improve both the mRNA technology and its delivery systems.
"The unique thing about the mRNA is because the integrity is so high is that your establishment rate is nearly 100 percent," Hamilton adds. "With mRNA, you can get them out, they establish efficiently and within five to six passages you can bank them, and they're integration-free, virus- free, DNA-free."
Other groups are also joining the mRNA bandwagon.  
At the recent International Society for Stem Cell Research (ISSCR) conference in Boston, Anton McCaffrey and colleagues at TriLink BioTechnologies presented a poster in which they discussed their efforts to improve mRNA reprogramming efficiency by modifying the mRNA molecules with pseudouridine and 5-methylcytidine. Previous research suggested that these substitutions would reduce the innate immune response to the mRNA molecules.  
The researchers found that the modified mRNAs not only induced high expression within a variety of reprogrammed cells, but also exhibited low toxicity with no sign of genomic modification. As well, the resulting iPSCs were able to differentiate into all three cell types.  
Not all groups have given up on non-integrative DNA-based reprogramming, however, as evidenced by the work of CDI, which still uses plasmids as part of its iPS 2.0 platform. The plasmids carry multiple reprogramming genes that alter gene expression within the cells, but do not themselves integrate into the genome.
Regardless of the method for reprogramming, Hamilton warns, the road for differentiating those reprogrammed cells into distinct tissues is not as clear.
"Differentiation is not at the point where reprogramming is," he opines. "With reprogramming, most of the protocols have been established and people are just working out the details, whereas there is a lot to be done yet with differentiation."
"People believe that once I have the iPSC from a given individual, I'm done," complains Parker. "What it doesn't address is really the hard part; it is relatively easy to produce an iPSC. We provide a kit to customers to do that in their own laboratories. It is another thing to produce highly pure populations in scale and in quality and quantity of a target tissue of interest."  
According to recent research, the starting material can have a lot to do with success in differentiation.  
Lined up
Although, in principle, stem cells can generally differentiate into any lineage, it appears that not all stem cells are created equal and different human embryonic stem cell (hESC) and iPSC lines can be biased toward differentiation into endoderm, mesoderm or ectoderm cell lineages. Unfortunately, this means that researchers may have to take time to test their stem cell isolates to see how they bias.
"It's like athletes and sports," explained Yi Zhang of Boston Children's Hospital in discussing research into hESCs. "Some athletes are built for football, some for baseball, some for swimming. Every cell line has its own strengths, and the challenge is knowing what those strengths are."
Zhang and colleagues may have found a shortcut in this situation using biomarkers. Publishing their findings in Stem Cell Reports, Zhang and Wei Jiang noted that hESCs that strongly express a gene called WNT3 seem to be heavily biased toward endoderm. Turning this observation on its head, they then found that by manipulating WNT3 gene expression—turning expression up or down—they could make hESCs more or less likely to differentiate into endoderm.  
Exactly how this happens is still a mystery, but Zhang believes this could be true of other biomarkers.
"We would like to find other markers and develop a scoring system," he says. "There are many hESC and iPSC lines, and we need a simple way to tell which to use in order to produce particular cell types."  
Aside from its own discovery efforts, CDI sees its customers as an opportunity to develop new cell lines, relying on Centers of Excellence agreements with companies like GSK and AstraZeneca.
"We produce cells that cross the silos that are typically constructed within the pharmaceutical industry," says Parker.  
By entering the industry through the safety and toxicity wing of pharma, he explains, CDI was able to show proof- of-concept with their cell lines, and that opened the doors to the R&D wing.
"Once the therapeutic areas saw that those cells worked in that safety area, they wanted to apply them in the discovery area, in screening applications," he says. "The Center of Excellence concept really evolved from being able to put an umbrella over the organization and then being able to provide all of our product offerings and some of the R&D resources that we have to deliver across their organization, so they could focus on the utilization of these materials, as opposed to trying to make them themselves."  
This work allows CDI to co-develop cell types of interest for a given customer such that, once CDI has met the client's requirements, they have the opportunity to commercialize those cells to other customers.
"It's a way of stimulating our product development pipeline with an outside partner who is going to be a customer once we complete that project," Parker says.  
CDI's projects rely on blood draws, one of the most common methods of harvesting cells, but hematopoietic cells can have drawbacks both physiologically and technologically, according to Stemgent's Hamilton.  
"This year, one of the limitations we had with mRNA reprogramming was delivery to blood-derived cell types," he says.  
To address this challenge, the company has focused its attention not on hematopoietic cell types, but rather on the rarer circulating endothelial progenitor cells (EPCs), which Hamilton says have unique properties that make them particularly valuable to stem cell research.
"One of them is that you can isolate them clonally," he explains. "When you talk about fibroblasts and do CD34 isolations from blood, you get a very heterogeneous genetic background."
The need to sort through a mosaic of genomic and proteomic differences, he says, can be very challenging.  
"The other thing is that EPCs don't carry the somatic mutations associated with blood disorders," he notes.
He points to two recent publications about EPC reprogramming that suggested these cells have a very low propensity to accumulate genetic insertions or deletions.
"Typically with fibroblasts, you accumulate copy number variations (CNVs)," he explains. "Thus, 80-plus percent of fibroblast lines are going to have a high degree of CNV accumulation just in going from fibroblast to iPSC."  
The situation is practically the reverse, however, when moving to EPCs.  
"When you reprogram EPCs to iPSCs—even with retrovirus, which is obviously not your ideal—80-plus percent have no accumulation of CNV," he says.  
Thus, he argues, you can be much more confident about the safety and fidelity of the resulting cell lines.
Code: E081329



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