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NYSCF Publishes Details on Automated Global Stem Cell Array Production Process


NEW YORK (GenomeWeb) – The New York Stem Cell Foundation has offered details on its $25 million project to automate the production of induced pluripotent stem cells (iPSCs) and create "populations-on-a-chip."

A paper in Nature Methods, published August 3, details the steps in the industrial-scale production of cells for NYSCF's Global Stem Cell array, a product that has already attracted numerous collaborators and could speed up research on genetic diseases and drug development.

"This is Affymetrix for stem cells," NYSCF CEO Susan Solomon told GenomeWeb. "This is going to allow clinical trials in a dish."

The Global Stem Cell Array starts with a bank of patient samples and ends with a multi-well plate full of patient-specific, differentiated, adult cells, with only several hundred steps in between. The process to reprogram and differentiate cells — created by the efforts of dozens of biologists, engineers, and software developers — is entirely driven by robotics, eliminating variability introduced by human hands. Reduced variability, along with the ability to put stem cells from a different patient in each well, could help scientists use the arrays to tease out differences in genetics that contribute to disease.

"We're interested in understanding how populations of people are affected by different diseases. Being able to derive multiple lines in parallel using standardized procedures allows us to make those comparisons," said Scott Noggle, NYSCF VP of stem cell research. "This is particularly important in diseases where there is a genetic component, but that genetic component is more of a risk factor than a single-gene mutation that is causative in the disease," including late-onset Alzheimer's disease and Parkinson's disease. "What we're trying to connect is that genetic defect to actual function of that gene in the disease process."

Collaborations with pharmaceutical companies and large research institutions, as well as disease-specific research foundations, could lead the Global Stem Cell array to become a widely-adopted tool for research.

Removing variability from the iPSC production process is what will allow such research into subtle effects of genes on disease. "There's a real lack of standardization in the field," Noggle said. "There's 50 to 100 ways of deriving the cell lines, another 50 to 100 of growing them. That's one of the major issues that we were trying to address with the automation, to provide standardization so that each cell line you derive can be faithfully compared to the next."

While developing the production process, the NYSCF scientists discovered wide variability in iPSC lines derived using manual processes. By automating the process, "we can really get a huge reduction in signal to noise so that you can understand what real biological differences are, as opposed to the differences from human hands in the process," Solomon said. This is especially important for a field recently suffering from problems with reproducibility. Noggle added that much of the variation comes about unconsciously, and that automation could reduce variation in the cell lines by as much as a third. "This is really important for when we start to look for subtle phenotypes in the cell lines in different disease cases," he said.

The Global Stem Cell Array project also helps address what Solomon saw as a lack of diversity in iPSC lines. "When we started thinking about this in 2008, we wanted to be able to look at problems at scale and understand how diversity in the population factored into the diversity in how people experience diseases," she said, noting that stem cell lines did not represent a diverse set of people, and the ability to generate human iPSCs had only been established in 2007. "We thought, 'It would be amazing if we had huge number of cell lines that were all identical that represented the diversity of world populations.'"

Those were, of course, tumultuous economic years, and Solomon said NYSCF applied for several grants to fund development of the project, but was denied. Undeterred, NYSCF turned to philanthropic sources for funding, and by 2010 seed funding for the project came together. Four years and more than 100,000 lines of computer code later, the Global Stem Cell Array became a reality.

One of the biggest challenges was designing a system that could be flexible to rapid changes in a nascent field. A panel of senior scientists evaluated state-of-the-art protocols while attempting to build in the capability to continually optimize the process, Solomon said.

The process itself starts with patient samples. Solomon said that NYSCF has created a recruiting "ecosystem" to obtain samples involving its own human subjects research team, disease-focused researchers and foundations, and the National Institutes of Health Office of Rare Diseases Research, among others.

"The first stage is creating a bank of cells from that tissue sample," Noggle explained. Often it's a skin sample from which the process isolates fibroblast cells that NYSCF will hold on to for the long term. "We have a starting bank of adult-type skin cells we can go back to as new methods improve for deriving stem cell lines," he said.

The next step is the crucial reprogramming stage, where genetic factors are introduced to the cells to turn back the clock on cell development and return the cells to a pluripotent state. To do so, NYSCF is using modified mRNAs, although it experimented with Sendai virus. "It allows us to standardize the reagents used during the reprogramming so we can consistently and reliably make the mRNAs for different transcription factors," Noggle said. "As a tool in manufacturing, it's a very reliable reagent that we found improved our ability to consistently generate the stem cell lines. Initial versions of [Sendai] virus were very finicky and difficult to standardize. Some of the more recent versions have improved, but we still prefer to use modified mRNA."

In the Nature Methods paper, the authors reported reaching reprogramming efficiencies between .001 percent and .16 percent per cell, or between one and 160 iPSCs per 100,000 cells processed, but Noggle said they've vastly improved the process since the study was submitted and it's now about five times more efficient. More importantly, the process can produce iPSCs from just about any patient sample, provided there isn't a genetic defect that prevents the reprogramming process, he said. Prior to automation, reprogramming results varied wildly and it was impossible to guarantee that a given sample would yield iPSCs.

After reprogramming, a purification step sorts out those that made it from those that didn't. The cells are harvested and sorted onto 96-well plates to go through quality control and ensure they're iPSCs. Cells are compared to embryonic stem cell lines and those that are bona fide are grown into a master stock of cells.

Finally, the cells are differentiated into the cell types NYSCF's partners are interested in studying. What makes an iPSC line turn into a cardiomyocyte versus a dopaminergic neuron? "Different food," Solomon said. Noggle added that the process leverages different signaling pathways, growth factors, and small molecules to direct iPSCs to differentiate into a number of adult cell types.

At the moment, NYSCF can create plated stem cell arrays of cardiomyocytes, beta cells, and several different kinds of neurons including those affected in Parkinson's disease; soon to follow are other neuronal subtypes, such as those involved in Alzheimer's disease and psychiatric disorders, and hepatocytes for toxicity screening.

Solomon described the Global Stem Cell Array project as a "moon shot," an apt description given that it relied more upon process engineering than new science. Still, Noggle said that there were some new discoveries made along the way, especially when it came to the reprogramming stage.

"We found that growth rates of fibroblasts in a particular media were critical for the efficiency of reprogramming the cells," he said.

Going forward, Solomon said that NYSCF is evaluating how it will distribute the stem cell arrays, but noted that for the time being it makes more sense to expand their existing capacity and operate under a "center of excellence" model, rather than build out multiple instances of the manufacturing process.

"This is not a vending machine, you can't just walk up to it and put in a quarter and you get your sample," Solomon said. "It requires a team of engineers and biologists to actually run this. What you get is a better canvas as opposed to something where you can operate it with reduced expertise."

The Global Stem Cell Array project already has a long list of collaborators including academic institutions like the Broad Institute; research groups studying Alzheimer's, Parkinson's, diabetes, Charcot-Marie-Tooth, and autism; and pharmaceutical companies including Pfizer, Roche, Takeda, and Novartis.

Research on drug development for rare sub-populations could be a killer application for the stem cell array, the NYSCF researchers suggested. "Our collaborators on the pharma side really believe this is going to speed up biomedical research and development in the most complex and expensive phase," she said. People could avoid potentially toxic side effects by providing tissue samples from which differentiated cells could be made. "We can do extensive testing on basically their avatars," Solomon said.

"This has particular value in drug development by allowing you to fail faster, reducing cost by allowing you to understand characteristics across populations that would be much too expensive for a clinical trial," Noggle added. "If you have a phenotype representative of a disease, if you take a population that has it and a population that doesn't and apply a drug that might affect that disease process in cell, you could understand whether that has an effect on an entire population or on a sub-population."

Solomon hopes that the stem cell arrays could help usher in personalized treatments. "You cannot do personalized one-on-one medicine unless you have a look at the biology of large numbers of people, sort them into different genetic buckets and then to, in effect, tease out what side effects which groups are going to exhibit and what the utility or efficacy of a drug is," she said.

Drug development and disease research may be the most obvious applications in the short term, but there could soon be others. Noggle suggested that stem cell arrays could be developed into diagnostic tools. Solomon left the door open even wider.

"We've thought of many of [applications]," Solomon said. "But many of our colleagues will think to use the arrays in ways we haven't even thought of."