Safer Culture Conditions for Stem Cells

Jeanne Loring from the Scripps Institute is the senior author of a very important study that examined the culture conditions for pluripotent stem cells.

Several scientists have discovered that induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) can accumulate cancer-causing mutations when grown in culture for extended periods of time (for example, see Uri Weissbein, Nissim Benvenisty, and Uri Ben-David, J Cell Biol. 2014 Jan 20; 204(2): 153–163). However, some laboratories have managed to keep ESCs in culture for extended periods without observing instabilities.

To try to tease apart why this might be the case, Loring and her group examined various culture methods and determined that some stem cell culture methods are associated with increased incidence of mutations in the DNA of stem cells.

“This is about quality control; we’re making sure these cells are safe and effective,” said Loring, who is a professor of developmental neurobiology at Scripps Research Institute (SRI) in San Diego, CA.

All cells run the risk of accumulating mutations when they divide, but previous research from Loring and her colleagues showed that particular culture conditions could potentially select for faster growth and mutations that accelerate growth. Such growth-enhancing mutations are sometimes associated with tumors.

“Most changes will not compromise the safety of the cells for therapy, but we need to monitor the cultures so that we know what sorts of changes take place,” said Ibon Garitaonandia, who is a postdoctoral research fellow in Loring’s laboratory at SRI.

New research from Loring’s group has shown how particular culture conditions can reduce the likelihood of mutations. Loring and her colleagues tested several different types of surfaces upon which the cells were grown. They also used different ways of propagating or “passaging” the cultures. When cells are grown in culture, the culture dishes must be scraped to get the cells off them and then the cells must be transferred to a fresh culture dish. How you do this matters: do you use enzymes to detach the cells, or do you mechanically scrape them off? Other culture techniques use layers of “feeder cells” that do not divide, but are still able to secrete growth factors that improve the health of the growing stem cells.

Loring and her crew tested various combinations of surfaces, passaging methods and feeder cell populations and grew the cells for three years with over 100 passages. Over the course of this experiment, the cells were sampled and analyzed for the presence of new mutations in their genomes.

It turns out that stem cells grown on feeder cells that are passaged by hand (manually) show the fewest growth-enhancing mutations after being cultured for three years.

Loring’s study also demonstrated the importance of monitoring cell lines over time. In particular, deletion of the TP53 gene, a tumor suppressor gene, in whose absence cancer develops, should be closely watched.

“If you want to preserve the integrity of the genome, then grow your cells under those conditions with feeder cells and manual passaging,” said Loring. “Also, analyze your cells. It’s really easy, she added.

When Thomson made the first human ESC lines, he used feeder cells derived from mouse skin cells.  However, the use of animal materials to make ESCs might pollute them with animal viruses and specific sugars from the surfaces of the animal cells might also contaminate the surfaces of the ESCs, making them unsuitable for regenerative medicine (see Stem Cells 2006; 24:221-229).  To address this problem, several laboratories have made “Xeno-free” ESC lines that were made without touching any animal products.  Some of these Xeno-free lines were made without feeder cells (see C. Ellerström, et al., Stem Cells. 2006 Oct;24(10):2170-6)., but others were made with human feeder cell lines (see K Rajala, et al., Hum Reprod. 2007 May;22(5):1231-8). Therefore, it appears, that the use of human feeder cell lines are preferable to feeder-free systems, given Loring’s findings.  However, it is also possible that such culture systems are also preferable for iPSCs, which do not have the problem of immunological rejection for patients, and do not require the killing of the youngest members of humanity.  Therefore, Loring’s work could very well benefit iPSC cultures as well.