Model citizens

Stem cells’ promise of disease-in-a-dish opens doors to truly personalized medicine

Randall C Willis

Ralph sits anxiously in an examination room. A few monthsago, he was diagnosed with rheumatoid arthritis, just the latest in a string ofconditions he gets to worry about.
His doctor did a blood draw and sent the samples off fortesting. Today, he learns the results.
"Good news, Ralph. We seem to have identified a drug thatshould help with your RA without interfering with your insulin regimen orincreasing 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 cellresearch suggest the day may not be too far down the road when a patient's owncells can be used to screen for drugs that work best for his or her conditionin the context of comorbidities and concomitant medications.
Open doors
When stem cell work was once something of a niche, offeringincredible therapeutic potential under an ethical cloud, the advent of inducedpluripotent stem cells (iPSCs) and discovery of a variety of stem cell typesfrom adult tissues has blown the doors off this field and generated buzz innumerous research areas. But buzz does not expertise make, and many researchersare finding their ideas overstep their abilities, opening the door forcompanies like Life Technologies Inc. (LTI).
"This is a very new field, and we thought people probablyaren't going to get a lot of expertise, resources or time, so this might alsobe an area they would outsource," says Carolyn Pettersson, senior manager ofLTI Discovery Services.
To address those needs, the company recently initiated astem cell service called CellModel.
"We tie a lot of it back to drug discovery, because webelieve that people will want to create different models and cell types and doassay development and screening, and that has been our focus and our strengthin services," Pettersson says.
"One aspect of someone coming to us could be to give uspatient samples, which we could sequence using our reagents," adds LTIAssociate Director David Piper. "By using these cells, you can get a better understandingof genetic variability as well as the disease state that the samples may comefrom. We can then take those samples and reprogram and differentiate them tospecific lineages, and again, using a multitude of fluorescent detectionsystems that we have from our Molecular Probes site, build assays and returnthe data. For a small company to do that is a big licensing commitment, a biginfrastructure commitment to start."
As Pettersson further explains, because CellModel isdeveloped using LTI's own products and instruments, the client can be trainedon the protocols and take over the process at any point, knowing it hascomplete access to all of the equipment and supplies they need—a one-stop shopsolution, as she describes it.
For Chris Parker, vice president and chief commercial officeof Cellular Dynamics International (CDI), which offers an array of cell typesthrough its iCell portfolio, it's a question of build or buy.
"Do you try to make your own picks and shovels, or do youbuy a pick and shovel and start mining for gold?" he asks. "We've found thatour customers are better suited and demonstrate more value by finding ways touse 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 thematerials such that we could truly manufacture these things consistently," heexplains. "So, if I was a researcher using a cardiomyocyte model, and you werea researcher using cardiomyocytes in New York, with cells from CDI, we couldcompare our results. Otherwise, if you made your cardiomyocytes and I made mycardiomyocytes, it's highly likely that they're not going to be the same."
This is going to be critical for using this data in aregulated environment, he adds, as organizations like the U.S. Food and DrugAdministration (FDA) will want to know whether the results are true across theboard.
An added feature, from Piper's perspective, is that stemcells offer researchers the best of all worlds because they offer theopportunity to perform experiments with the ultimate control, the patient.
Traditionally, experimental controls have consisted of alarge diversity of individuals who do not have the disease of interest, andcomparing them to the people with disease, he explains. "In the best-casescenario, perhaps those people are related. But another fundamental way ofapproaching that is to take a disease sample—say you have a monogenicdisease—and use a technology like TAL editing or something along those lines toaddress a single nucleotide in the genome. You change it to make the cellwild-type, and now you have a wild-type control that is genotypically the sameexcept for that single monogenic disease."
It is tantamount to using an identical but healthy twin tounderstand what has gone wrong in the unhealthy twin and upon whom to testtreatments.
CDI's Parker concurs.
"We can use that same genetic engineering methodology notonly to correct and create what we call isogenic controls, but we can alsomodify the genome," he suggests. "We can do over-expression to create a diseasemodel within a normal patient and compare it to a disease model from an iPSCpatient."
Among the molecular tools CDI has licensed are LTI's GeneArtPrecision TALs (TALENs) and Sigma's CompoZr ZFN technologies, in dealsannounced in June.
Of course, reprogramming an adult cell into an iPSC is notalways straightforward.
Integrating non-integration
One of the challenges of developing stem cell lines is notchanging the genetic background of the cell while returning it to a pluripotentstate, a problem exacerbated by integrative vectors such as most viruses.
"You want to avoid viruses in general, which are notgenerally looked at highly by the FDA," says Brad Hamilton, director of R&Dat Stemgent. "For reprogramming, there are some DNA vehicles—plasmids,episomes, minicircles or things like that—which you could call a transientstate, 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 theirfuture use as either a research tool or therapeutic (more on that next month).
"We'd obviously seen publications for Sendai and episomesand for other DNA systems, but it wasn't quite what we felt was going to be theultimate 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 Rossiand Luigi Warren, leaders in the development of mRNA for cellularreprogramming, to improve both the mRNA technology and its delivery systems.
"The unique thing about the mRNA is because the integrity isso 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 tosix 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 TriLinkBioTechnologies presented a poster in which they discussed their efforts toimprove mRNA reprogramming efficiency by modifying the mRNA molecules withpseudouridine and 5-methylcytidine. Previous research suggested that thesesubstitutions would reduce the innate immune response to the mRNA molecules.
The researchers found that the modified mRNAs not onlyinduced high expression within a variety of reprogrammed cells, but alsoexhibited low toxicity with no sign of genomic modification. As well, theresulting iPSCs were able to differentiate into all three cell types.
Not all groups have given up on non-integrative DNA-basedreprogramming, however, as evidenced by the work of CDI, which still usesplasmids as part of its iPS 2.0 platform. The plasmids carry multiplereprogramming genes that alter gene expression within the cells, but do notthemselves integrate into the genome.
Regardless of the method for reprogramming, Hamilton warns,the road for differentiating those reprogrammed cells into distinct tissues isnot as clear.
"Differentiation is not at the point where reprogrammingis," he opines. "With reprogramming, most of the protocols have been establishedand people are just working out the details, whereas there is a lot to be doneyet with differentiation."
"People believe that once I have the iPSC from a givenindividual, I'm done," complains Parker. "What it doesn't address is really thehard part; it is relatively easy to produce an iPSC. We provide a kit tocustomers to do that in their own laboratories. It is another thing to producehighly pure populations in scale and in quality and quantity of a target tissueof interest."
According to recent research, the starting material can havea lot to do with success in differentiation.
Lined up 
Although, in principle, stem cells can generallydifferentiate into any lineage, it appears that not all stem cells are createdequal and different human embryonic stem cell (hESC) and iPSC lines can bebiased toward differentiation into endoderm, mesoderm or ectoderm celllineages. Unfortunately, this means that researchers may have to take time totest their stem cell isolates to see how they bias.
"It's like athletes and sports," explained Yi Zhang ofBoston Children's Hospital in discussing research into hESCs. "Some athletesare built for football, some for baseball, some for swimming. Every cell linehas its own strengths, and the challenge is knowing what those strengths are."
Zhang and colleagues may have found a shortcut in thissituation using biomarkers. Publishing their findings in Stem Cell Reports, Zhang and Wei Jiang noted that hESCs that stronglyexpress a gene called WNT3 seem to be heavily biased toward endoderm. Turningthis observation on its head, they then found that by manipulating WNT3 geneexpression—turning expression up or down—they could make hESCs more or lesslikely to differentiate into endoderm.
Exactly how this happens is still a mystery, but Zhangbelieves this could be true of other biomarkers.
"We would like to find other markers and develop a scoringsystem," he says. "There are many hESC and iPSC lines, and we need a simple wayto tell which to use in order to produce particular cell types."
Aside from its own discovery efforts, CDI sees its customersas an opportunity to develop new cell lines, relying on Centers of Excellenceagreements with companies like GSK and AstraZeneca.
"We produce cells that cross the silos that are typicallyconstructed within the pharmaceutical industry," says Parker.
By entering the industry through the safety and toxicitywing of pharma, he explains, CDI was able to show proof-of-concept with theircell lines, and that opened the doors to the R&D wing.
"Once the therapeutic areas saw that those cells worked inthat safety area, they wanted to apply them in the discovery area, in screeningapplications," he says. "The Center of Excellence concept really evolved frombeing able to put an umbrella over the organization and then being able toprovide all of our product offerings and some of the R&D resources that wehave to deliver across their organization, so they could focus on theutilization of these materials, as opposed to trying to make them themselves."
This work allows CDI to co-develop cell types of interestfor 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 pipelinewith an outside partner who is going to be a customer once we complete thatproject," Parker says.
CDI's projects rely on blood draws, one of the most commonmethods of harvesting cells, but hematopoietic cells can have drawbacks bothphysiologically and technologically, according to Stemgent's Hamilton.
"This year, one of the limitations we had with mRNAreprogramming was delivery to blood-derived cell types," he says.
To address this challenge, the company has focused itsattention not on hematopoietic cell types, but rather on the rarer circulatingendothelial progenitor cells (EPCs), which Hamilton says have unique propertiesthat make them particularly valuable to stem cell research.
"One of them is that you can isolate them clonally," heexplains. "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 proteomicdifferences, he says, can be very challenging.
"The other thing is that EPCs don't carry the somaticmutations associated with blood disorders," he notes.
He points to two recent publications about EPC reprogrammingthat suggested these cells have a very low propensity to accumulate geneticinsertions or deletions.
"Typically with fibroblasts, you accumulate copy numbervariations (CNVs)," he explains. "Thus, 80-plus percent of fibroblast lines aregoing to have a high degree of CNV accumulation just in going from fibroblastto iPSC."
The situation is practically the reverse, however, whenmoving 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 thesafety and fidelity of the resulting cell lines.

Randall C Willis

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