LA JOLLA, Calif.—In what they are calling "the mostcomprehensive study of human pluripotent stem cell variation to date," a teamof researchers from the Scripps Research Institute and the University ofCalifornia, San Diego, has discovered dynamic changes in gene regulation inthese cells, a finding that may have important implications for using them forbasic and clinical research.
Human pluripotent stem cells (hPSCs) can give rise tovirtually every type of cell in the body, and thus hold huge potential for cellreplacement therapies and drug development. But there is still much to belearned about how these cells behave in the lab, says Prof. Jeanne Loring ofScripps Research, a lead author on a study recently published in the journal Cell Stem Cell.
Loring's lab focuses on basic and translational researchapproaches to understanding stem cells at a molecular level, and its scientistsare working to apply that knowledge in the field of regenerative medicine. Lastyear, the team reported recurrent changes in the genomes of hPSCs as they areexpanded in culture, and their development of a technique for purifying cellmixtures.
"One of the big issues about using human pluripotent stemcells for any kind of clinical application—including disease studies and drugdevelopment—is that these cells are unlike cells anyone has ever studiedbefore, and we must learn more about them. The first thing that came to mindfor us is that these cells are like cancer cells in some ways. If one cell hasa slight survival advantage over others, as it grows and divides, this cellwill take over the culture. The same thing happens in cancers, especially bloodcancers."
The team used tiny beads to attach lectin to stem cells. Thecells that washed past were almost all non-stem cells, and the scientistsobserved that both cell types could be collected separately for use in researchor in treatments. This work presented a new way to solve purification andsafety problems in stem cell research, says Loring.
"But we didn't answer the question of whether these changesare close to the genes involved in maintaining those cells as beingpluripotent," she notes. "The changes that occurred in culture were enhancingthat cell type. That may or may not be a problem, but what is important is thatwe pointed out that it happened."
Now, in a follow-up study that appears in the May 4 issue ofCell Stem Cell, Loring's lab isreporting that these cells can also change their epigenomes, the patterns ofDNA modifications that regulate the activity of specific genes. These changesmay influence the cells' abilities to serve as models of human disease anddevelopment.
Specifically, the team assessed the state of both DNAmethylation and gene expression in more than 200 hPSC samples from more than100 cell lines, along with 80 adult cell samples representing 17 distincttissue types. Both DNA methylation and demethylation are important regulatoryprocesses in cellular differentiation.
Key to the research was a new global DNA methylation array developedin collaboration with Illumina Inc. that detects the methylation state of450,000 sites in the human genome. The results showed surprising changes inpatterns of DNA methylation in the stem cells. Because of the unprecedentedbreadth of the study, the researchers were able to determine the frequency ofdifferent types of changes.
The team observed that pluripotent cells differ from somaticcells at sites in the genome that are generally considered to be epigeneticallystable—the inactivated X chromosome in female cells and imprinted loci. Xchromosome inactivation (XCI) was not erased following the reprogramming ofhuman fibroblasts, but was lost over time, leading to a loss of dosagecompensation of subsets of X chromosome genes. This includes a large number ofX-linked disease genes, which may complicate a large number of hPSC-basedmodels of X-linked disease. Aberrations in genomic imprints are frequent inhPSCs, and all of the hPSCs analyzed in this study had abberant DNA methylationof at least one imprinted gene.
"There are whole families of diseases associated withabnormal imprinting," says Loring. "We're probably going to find that in a lotof cases, neurodevelopmental diseases will have abnormalities in methylationthat we can trace to a mutated gene, and then use that as leverage to try tofigure out how they are controlled."
Some diseases or conditions Loring specifically mentions areRett's syndrome, Fragile X syndrome and even autism.
"In the autistic brain, this would be a handy way to explainwhy you have certain characteristics," she adds.
The team is now working on controlling the imprinting andwhich genes are turned on or off when cells differentiate.
"These are not small questions, but now we know that we needto ask them," says Loring.
The results presented by her team are interesting from adevelopmental biology perspective because "it places us on the edge ofunderstanding the development of some diseases," Loring points out.
"When you think about embryonic development, it all goes sowell, and it is hard for us to understand how all of these things can fall intoplace," she says. "This research will give us insight into how that happens.This is the kind of thing we couldn't have done a year ago because we didn'thave the tools. I feel like we are in an exciting place right now because we'regetting really good at culturing cells and doing bioinformatic analysis. Thecombination of these things is leading to interesting kinds of insights thatwouldn't have been possible before."
The study, "Recurrent Variations in DNA Methylation in HumanPluripotent Stem Cells and their Differentiated Derivatives," was supported bythe California Institute for Regenerative Medicine and the U.S. NationalInstitutes of Health. Loring's colleagues on the multi-site project includedGulsah Altun, Candace Lynch, Ha Tran, Ileana Slavin, Ibon Garitaonandia,Franz-Josef Müller, Yu-Chieh Wang, Francesca S. Boscolo and Eyitayo Fakunlefrom Scripps Research; Julie V. Harness and Hans S. Keirstead from theUniversity of California at Irvine; Mana M. Parast from the University ofCalifornia San Diego; Tsaiwei Olee and Darryl D. D'Lima from Scripps Health; BiljanaDumevska and Andrew L. Laslett from the Commonwealth Scientific and IndustrialResearch Organization in Australia; Uli Schmidt from the Stem Cell Laboratoryin Sydney, Australia; Hyun Sook Park and Sunray Lee from the Laboratory of StemCell Niche in Seoul, South Korea; Ruslan Semechkin from International Stem CellCorp.; and Vasiliy Galat from Children's Memorial Research Center in Chicago.
