NEW YORK (GenomeWeb News) – A new microarray-based approach is proving useful for detecting unique microRNA patterns in human embryonic stem cells.
An international team of researchers, including scientists from the Scripps Research Institute and San Diego-based company Illumina, used a method similar to Illumina’s DASL gene expression assay to compare more than a dozen stem cell lines with several other cell types. The results appeared in the online edition of the journal Stem Cells yesterday.
“We report the most comprehensive determination of microRNA expression in human embryonic stem cells to date and detail differences in microRNA expression that may be key to regulating stem cell pluripotence — the ability to change into other cell types,” senior author Jeanne Loring, founding director of the Scripps Research Institute’s Center for Regenerative Medicine, said in a statement.
As well as being able to become any type of cell, embryonic stem cells can regenerate themselves indefinitely. In an effort to characterize the molecular regulatory processes that give human embryonic stem cells these properties, Loring and her team focused on microRNAs — small, non-coding RNAs that bind to and regulate messenger RNAs.
This is not the first effort to determine the role of miRNAs in stem cells. Previous research in mice suggests miRNAs may contribute to stem cells’ ability to self-renew and differentiate. And, Loring and her colleagues noted, a 2007 study outlined the sequencing of more than 300,000 miRNA clones from murine stem cells. But, they say, the design and research tools used in previous studies make it difficult to draw conclusions about miRNA expression in stem cells.
“The unique biology of miRNAs, as well as limitations in detection and quantitation methods for these small RNAs, has made it difficult to understand their functions in higher animals,” the authors wrote. “Furthermore, these are classes of closely related, but not identical miRNAs that differ at only one or a few nucleotides.”
For this paper, though, the researchers used a new microarray-based approach that allowed them to do rapid, reproducible testing of miRNA content and expression in 26 different cell types. The technique uses a modification of Illumina’s high-throughput, gene expression profiling, cDNA-mediated Annealing, Selection, Extension, and Ligation (DASL) assay.
The team used 700 assay probes corresponding to 397 annotated human miRNA sequences from a 2006 Sanger database and 303 human miRNAs recently discovered in human and chimpanzee brain samples.
When they compared miRNA expression in human embryonic stem cells with other cell types, Loring and her team found 150 miRNAs that were expressed at different levels in stem cells also clustered together in the genome — 76 were up-regulated, while 74 were down-regulated in these stem cells.
Intriguingly, several miRNAs that are associated with cancer were more highly expressed in the stem cells. On the other hand, miRNAs that are known as tumor suppressors appear to be relatively uncommon in embryonic stem cells.
“[W]hile oncogenic miRNAs are highly expressed in some human embryonic stem cells, the cells themselves are not oncogenic,” Loring said. “This might be a cautionary tale, though, with the moral that we pay particular attention to changes made in cellular microRNA so we don’t trigger any abnormal events.”
The researchers were also able to classify their cell lines based on miRNA spatial clustering and expression patterns as well as the miRNAs’ potential target mRNAs and targeting sequences.
“These findings significantly extend our knowledge, and suggest potential roles for miRNAs in regulating cellular pluripotence and self-renewal,” the authors wrote. “We conclude that miRNA profiling can be used for robust classification of diverse cell types, and that [human embryonic stem cells] possess a unique miRNA signature, with the up-regulated miRNA dominated by a single seed sequence.”
The results suggest it may be possible to unravel the functional roles of various miRNAs in stem cell maintenance and differentiation and, Loring suggested, to reprogram differentiated cells.