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BioTechniques
01/18/2012 Rachelle Dragani

An international group of scientists wanted to analyze stem cells from different ethnicities to advance the prospects of regenerative medicine. But what they found is that changes in one particular area of the genome may unite the stem cells of many different populations. Rachelle Dragani reports.

In 2009, Jeanne F. Loring had a dream. In her vision of the future, all ethnicities of the world would be represented in stem cell research. But at that time, Loring and colleagues struggled to find diversity in human embryonic stem cell (hESC) lines. In a paper published in Nature Methods in January 2010, her team found that most of the 47 commonly studied cell lines were derived from either European or Eastern Asian donors (1). Because no cell line that they genotyped was of African origin, they generated one to expand the diversity of stem cell lines available to researchers.

“There’s not a lot of value in making a new pluripotent stem cell line now unless it has something new to offer,” Loring said in a statement at the time. “I think that increasing ethnicity and genetic diversity is an important reason for generating new lines.”

In the end, the group decided to focus on 125 hESC lines. While about half of these cell lines still represented European ancestry, the remainder represented Asian, African, and Middle Eastern ancestries. Source: Nature Biotechnology

All things considered, Loring and colleagues highlighted that the lack of ethnically defined and ethically diverse stem cell lines will compromise drug screening and toxicity studies as well as the development of stem cell therapies. Specifically, this increased diversity would provide a greater understanding of genetic variation in human populations, producing more effective treatments for disease.
Two years later, a group of international stem cell researchers has taken another leap towards increasing our understanding of stem cell diversity. The International Stem Cell Initiative (ISCI) has screened 136 ethnically diverse stem cell lines, and, through their analysis, has discovered that some variation may be common to a good percentage of the world’s population.

The Initiative

At the University of Sheffield, Peter Andrews is co-director of the Center for Stem Cell Biology where he has been studying stem cell biology, specifically the adaptation of hESCs to in vitro culture. While Andrews understands that these cells have the potential to improve drug screening and the development of regenerative medicine, there’s a problem. We just don’t know enough about how hESC regulate self-renewal and differentiation.

For one thing, stem cells are complex little beasts, constantly mutating, which makes them difficult to study. In addition, these unidentified or misunderstood mutations in stem cells that are implanted into a patient to help grow back a missing organ could alter the lifecycle of a stem cell and wreak havoc on the patient’s body, causing more harm than help. In the end, genetic abnormalities that accumulate in cultured stem cells could make regenerative medicine treatments too risky.

To improve the understanding of stem cell biology, in 2005, Andrews helped establish the ISCI, an international consortium of laboratories that is helping to define standards in the field. By 2007, the group had published their first paper in Nature Biotechnology, reporting the characterization of 59 hESC lines from 17 laboratories around the world (2). As a result, the researchers found that even though these stem cell lines possessed different genotypes and were derived using different techniques, they expressed similar markers for stem cells. Furthermore, they found no evidence of contamination, a common problem for cell lines in general.

In 2010, the consortium continued their mission by evaluating defined feeder-free culture systems for the propagation of hESCs. In a paper published in In Vitro Cellular and Developmental Biology – Animal, five separate labs tested eight different cell culture methods using 10 different ESC lines (3). In the end, only two commercial media supported most cell lines for ten passages. The others failed for a variety of reasons, including lack of attachment, cell death, and cell differentiation.

When Loring and colleagues published their paper on the diversity of hESC lines in 2010, Andrews and the ISCI took notice. So, for one of their next initiatives, the consortium decided to explore the genomic variation of ethnically diverse stem cell lines.

Stem Cells of the World, Unite

One goal at the outset of the ISCI’s study was to include stem cells from as many ethnicities as possible. In the two years since Loring’s paper, the number and ethnic diversity of available stem cell lines has increased, providing the ISCI with a large and diverse sample set to analyze. Because the ISCI already had formed a network of international stem cell researchers, locating 39 research laboratories to collect and derive these diverse stem cell lines wasn’t all that difficult. In contrast, legal roadblocks and international shipping hiccups were a much larger problem when they were putting together this diverse collection of embryonic stem cells.

In the end, the group decided to focus on 125 hESC lines. While about half of these cell lines still represented European ancestry, the remainder represented Asian, African, and Middle Eastern ancestries. The cell lines were further characterized by population sub-groups, including Han Chinese, Italian, East African, and Central European ancestries.

Although these cell lines were ethnically diverse, they were all derived from hESCs from in vitro fertilization clinics, therefore most likely representing the wealthy of each country who had the resources to pay for such treatment. All in all, this was the most ethnically diverse sample set that the ISCI team could assemble, and they hoped it would provide some interesting insights into stem cell biology.

“Many of these studies, in a way, have got quite simple experimental goals, and really the essence of this study was that it was easily collaborative. It offers a template of how you can do this to other things, and now we look at a plan for differentiation,” said study author Paul Gokhale, an associate researcher in Andrews’ lab at the Center for Stem Cell Biology.

More to Come on Chromosome 20

Once the global samples were collected, researchers began analyzing the gene patterns through molecular karyotyping. As a result, the ISCI team found that most of these cell lines are simply normal, developing no harmful mutations, which can have a big impact on future treatments. Without having to account for complex mutations that will change during long-term culture, the paper helped researchers realize they can work with a relatively stable group of cells while attempting to create targeted treatments.

“The fact that of the 125 cell lines tested, over 65% of them exhibited normal karyotypes in long-term culture bodes well for the use of human embryonic stem cells for cell therapy in the future,” said Steve Oh, principal scientist at the Bioprocessing Technology Institute in Singapore.

To their surprise, the changes in one region of chromosome 20 were remarkably stable across the stem cell lines. In a paper recently published in Nature Biotechnology, the ISCI reported that of the hESCs analyzed, more than 20% of the cell lines acquired similar amplifications in the 20q11.21 region during extended culture (4). This area not only includes three ES-cell expressed genes but also has been linked to certain cancers.

Specifically, the amplicons gathered in a specific gene in that region, the gene BCL2L1. Recent research on this gene has linked it to a selective growth advantage during development in both stem cell biology and cancer biology. If the researchers could target a way to safely disrupt the expression of BCL2LI in cancer cells, these cells might not have such an easy time proliferating.

“We learned a lot of these lines stayed normal, relatively speaking,” says Gokhale. “So, a lot of these have been growing for a hell of a long time, given that you can look and see the actual population doubled, and yet many of the lines stayed absolutely normal. It’s good news because it means we’re going to be able to control this process a lot longer.”

References

Laurent LC, Nievergelt CM, Lynch C, Fakunle E, Harness JV, Schmidt U, Galat V, Laslett AL, Otonkoski T, Keirstead HS, Schork A, Park HS, Loring JF. 2010. Restricted ethnic diversity in human embryonic stem cell lines. Nat Methods. 7:6-7.
Adewumi O, Aflatoonian B, Ahrlund-Richter L, Amit M, Andrews PW, Beighton G, Bello PA, Benvenisty N, Berry LS, et al. 2007. Characterization of human embryonic stem cell lines by the International Stem Cell Initiative. Nat Biotechnol. 25:803-16.
Akopian V, Andrews PW, Beil S, Benvenisty N, Brehm J, Christie M, Ford A, Fox V, Gokhale PJ, Healy L, et al. 2010. Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells. In Vitro Cell Dev Biol Anim. 46:247-58.
Amps K, Andrews PW, Anyfantis G, Armstrong L, Avery S, Baharvand H, Baker J, Baker D, Munoz MB, et al. 2011. Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage. Nat Biotechnol. 29:1132-1144.