Stem cells undergo chromosomal changes after long periods in culture

A USP team participated in an international study analyzing 125 stem cell lines produced in 38 laboratories, recently published in Nature Biotechnology.

By Fábio de Castro

Agência FAPESP – Brazilian researchers participated in a large international study showing that when human embryonic stem cells were maintained in culture over a long period of time, the cells underwent changes in their chromosomal structure that could compromise their utility in therapeutic applications.

The study, which was the cover story in Nature Biotechnology’s December edition, analyzed 125 human embryo stem cell lines and 11 induced pluripotent stem cell lines. The objective of these analyses was to identify possible genetic changes in cells grown in culture over time.

The work was coordinated by the International Stem Cell Initiative (ISCI), which involves 38 laboratories throughout the world, including LaNCE, the National Embryonic Stem Cell Laboratory at the Universidade de São Paulo Biosciences Institute (IB-USP). LaNCE is coordinated by Lygia da Veiga Pereira.

The LaNCE team obtained the first line of stem cells to be developed in Latin America (BR-1) in 2008, samples of which were used in the ISCI study. A researcher in Pereira’s group, IB-USP doctoral student and FAPESP fellow Ana Fraga, also participated in the study.

According to Fraga, the karyotype, or set of chromosomes, in most of the lines remained stable in culture for some time. However, over a period of six months in culture, changes accumulated progressively, most commonly affecting chromosomes 1, 12, 17 and 20.

 “Not all the genes involved in these chromosomal changes are known. But we know that chromosome 20, for example, holds an important gene related to cellular death. From now on, it will be necessary to test the point at which these modifications could affect therapeutic applications,” Fraga told Agência FAPESP.

According to Fraga, it was already known that cultured cells are somewhat unstable, enough to compromise their therapeutic applications. However, this was the first robust study to confirm the chromosomal changes that occurred in cells that were maintained in culture for long periods.

 “The advantage of this study is that it brings together analyses of over a hundred lines of embryonic stem cells from different ethnic origins. All of these experiments were carried out in a very rigorous manner, under the same parameters, in the same laboratory, with the same research protocols and methodology,” she said.

Fraga explains that the DNA extracted from lines used around the world, including BR-1, was sent to Singapore for epigenetic analyses. One lot of frozen cells was sent to the United Kingdom for cytogenetic analysis.

It is possible that the cultivation conditions are causing the observed changes. “This hypothesis will be tested in future studies. The modifications, however, don’t necessarily mean that cultivation will result in nonviable therapeutic applications of the lines,” she said.

Application and effects

In cultivating human embryonic stem cells, scientists have “imitated” the methodology used for the analysis of murine (mouse) cell lines. What the most recent studies are showing, says Fraga, is that human cells do not behave exactly like the murine cells.

“When we work with an animal model, the variations may not make a great difference for the experiment. But in human cells, we need to get as close to in vivo conditions as possible, as the results will be applied in therapies,” she said.

“This is why there is concern about the changes we observed. But I don’t find the results alarming, nor do they mean that the therapeutic application of these cells will have deleterious effects. We merely confirmed that there is a need for more studies on the ideal conditions for the cultivation of embryonic stem cells,” she added.

The article "Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage," (doi:10.1038/nbt.2051) by Lygia Pereira, Ana Fraga and others can be read by Nature Biotechnology subscribers at www.nature.com/nbt/journal/v29/n12/full/nbt.2051.html.