It's estimated that there are between 2,000 and 2,400 active cell-therapy trials in progress worldwide. A slice of these involve (pluripotent or adult) stem cells that are induced to differentiate in vitro. It’s vital to know what these cells that are potentially introduced into humans are and what they do—to have information about their biology, history, development and clinical potential.
Here we look at some of the processes and experiments researchers are using to devise and validate novel cell-therapy research methods utilizing stem cell-derived cells.
No “always”
In cell-therapy manufacturing—unlike more traditional pharma or biotech manufacturing—there are few standard assays for characterizing the cells that compose the final product. "Regulators expect us to use the analytical methods that will best fit the job, and that's determined by the types of cells and the application," says Scott Burger, founder and principal at the Advanced Cell and Gene Therapy consultancy.
Characterizing the cells based on identity and purity, Burger says. "will be at the level of gene expression, and cell surface marker phenotype will be particularly important."
The same is largely the case in the research arena, as well, where a host of tools are at the lab’s disposal.
For Rick Cohen, director of the Stem Cell Training Course at Rutgers University, the process begins with making sure the cells are cytogenetically normal. This can be done by G-banding, multispectral probes or even a DNA microarray. “The cost of aCGH [array-comparative genomic hybridization], as compared to karyotyping, may be a little higher, but the resolution can be as low as 50 kb—that’s a lot smaller than megabases,” Cohen says. Though he cautions that “you can get a little paranoid, because some variation is normal.”
Cells that are differentiated, in theory should have lost their “stemness.” This would typically be verified by looking at the expression of key genes associated with pluripotency by PCR, or by immunocytochemistry (ICC). There should also be no evidence of any reprogramming agents such as Sendai virus or exogenous RNA. “When you don’t see them, then you can move on to the next stage, which is characterizing the lineage,” Cohen shares.
Lineage testing
The source of the cells—whether chimeric antigen receptor T (CAR-T) cells are made from induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs) or lymphocytes taken from peripheral blood, for example—should make no difference in term of how they are characterized.
Cells destined for cell therapy are most often differentiated to the desired phenotype, but “in some cases we administer cells that undergo further maturation after administration—it varies enormously, depending on the cell type and application,” Cohen explains. The function of every cell type, at every stage of development, is not always well understood.
“Cell surface analysis by flow cytometry is kind of the granddaddy of all the analytic methods we use—it’s been an absolutely fundamental,” says Burger. “Gene-expression arrays looking at RNA are increasingly important. We also test for potency — biological function — but that generally requires the most analytical development of all."
For many cell types, characterization kits are available for flow cytometry and ICC. “There are a lot of antibodies out there for a lot of these different markers, so we’ve done the work for the researcher and screened all of them. And we’ve put the best performing ones in a kit,” explains Nick Asbrock, product manager for pluripotent and differentiated cell technologies at MilliporeSigma.
It's not just markers for the differentiated cells that should be queried, but markers for intermediates in the process as well —in the final product, those would be considered impurities, and “we need to detect cells that haven't differentiated as far or in the way that was intended," says Burger. Monitoring process intermediates also helps refine and define the manufacturing process, he adds.
Other tools
Cell-therapy research sometimes includes following cells that have been introduced into an animal model or looking at cells that have been used to form organoids in a dish.
Immunohistochemistry (IHC) can provide context to the staining, indicating where cells have migrated in addition to what markers they express.
When antibodies are not available or are not sensitive or specific enough, RNA in situ hybridization (ISH) may be used for contextualized expression analysis. But that, too, can suffer from sensitivity or specificity issues, notes Courtney Anderson, senior scientist at Advanced Cell Diagnostics (ACD). Unlike traditional ISH, ACD’s RNAscope ® ISH system requires two probes to hybridize next to each other on the target RNA to assure specificity; the signal is then amplified through a cascade of events that allows for single-molecule detection sensitivity.
There are, of course, cell-therapy researchers who engage in other types of analyses. “What’s coming into vogue is doing some type of epigenetic testing,” says Cohen. While whole genome epigenetic analysis is really still out of reach for most labs, he explains, it may still be possible to follow changes in a few key genes that indicate, for example, that the cells are poised to become neurons.
For those “doing any type of clinical type research,” Cohen recommends identity testing of the cells; kits and services are available from several vendors. “Then test it every several passages to make sure you haven’t contaminated your cultures with a different culture.”
With so many different types of cells being explored for so many different cell-therapy applications—from repairing damaged tissue to destroying cancer—it’s no wonder there are so many ways to characterize them. Just make sure that they are what you want them to be, aren’t what they should no longer be, and that they do what they’re supposed to do.
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