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Scientists rid stem cell culture of key animal cells


Tackling a pressing and controversial technical barrier in stem cell biology, scientists at the WiCell Research Institute and the University of Wisconsin-Madison have crafted a recipe that allows researchers to grow human embryonic stem cells in the absence of mouse-derived "feeder" cells, long thought to be a source of potential contamination for the therapeutically promising cells.

Caption: This image depicts a colony of human embryonic stem cells grown over a period of 10 months in the absence of mouse feeder cells. The cell nuclei are stained green; the cell surface appears in red. Photo: courtesy Ren-He Xu

The new findings, appear today (Feb. 17) in the journal Nature Methods and come on the heels of a recent University of California study showing that existing stem cell lines are already contaminated with an animal molecule. The potential threat of animal pathogens tainting human stem cell lines poses a problem for the safe clinical use of many, if not all, of the current cell lines now in use.

Until now, scientists have had to grow and sustain stem cells through the tedious daily task of generating mouse feeder cells from mouse embryos. Feeder cells, or fibroblasts, are connective tissue cells that form the matrix upon which stem cells grow.

The mouse feeder cells were an important ingredient in the mix of culture materials required to keep stem cells in their undifferentiated "blank slate" state. Embryonic stem cells are capable of forming any of the 220 tissues and cells in the human body and, in culture, are constantly trying to migrate down different developmental pathways. Maintaining stock cultures in their undifferentiated state is critical.

The feeder cell dogma now can be overturned, says lead investigator Ren-He Xu, a senior scientist at WiCell, a private, nonprofit research institute. "This work completely gets rid of the need for feeder cells," says Xu. "It also significantly reduces the daily labor of preparing the feeder cell-conditioned medium."

"It is important that the culture of human ES cells be simplified so that the average scientist can use them without extensive prior training," says James Thomson, a UW-Madison professor of anatomy and a co-author of the Nature Methods paper. "This development is a good step in that direction. Also, clinically, the feeder cells were one of the main sources of potential contamination with pathogens, so their elimination should improve safety. However, not all the animal components have been removed from the media yet, but this is an important step."

Working with three of WiCell’s five human embryonic stem cell lines, Xu and his team explored the molecular interactions within the stem cell growth medium. He discovered that, in certain conditions, a protein known as fibroblast growth factor 2 (FGF2) accomplishes the same critical role that feeder cells are thought to play: ensuring that the stem cells remain in their undifferentiated state and capable of proliferation.

"We’ve got it down to the mechanism," Xu says.

Moreover, Xu made the surprising discovery that the very molecules that encourage human embryonic stem cells to differentiate appear to inhibit differentiation in mouse embryonic stem cells.

Aside from feeder cells, two other sources of animal material remain in stem cell culture materials. One, Matrigel, is a product that is essentially a plate-coating matrix of cells extracted from mouse tumors. Serum replacement, which is bovine in origin, is the other animal material still needed to culture stem cells.

Xu became interested in unveiling molecules derived from the mouse feeder cells because, in their absence, stem cells start to differentiate within two to three days. Xu started by evaluating the effect of changing stem cell growth conditions - using less feeder cell material, or no serum replacement, for instance.

Unexpectedly, Xu found that the presence of serum replacement promoted stem cell differentiation. Digging deeper, he found that serum replacement mimics the activity of bone morphogenetic protein (BMP), a molecule known to kick-start embryonic development, or in this case, cell differentiation.

If serum replacement triggers stem cell differentiation, Xu deduced, there must be feeder cell molecules that oppose BMP activity. Experiments confirmed this to be true.

Next, Xu elevated concentrations of FGF2, a protein routinely used for human embryonic stem cell culture, to test whether FGF2 preserves undifferentiated stem cells in the absence of BMP. The result was that "the cells looked perfect." Xu says he has grown the resulting stem cells in the desired undifferentiated state for almost a year.

Although the new work "dramatically reduces the possibility of contamination" from animal pathogens, Xu warns that the continued use of serum replacement and Matrigel means that contamination remains a concern. The ultimate goal, he says, would be to culture stem cells in media completely free of any animal products.

Ren-He Xu | EurekAlert!
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