The next players
Beyond cell imaging and gene expression, other applications of single stem cell analysis are hardly thick on the ground. No one carrying out proteomic or metabolomic studies on individual stem cells could be located for this article. Such research is typically done on whole organisms, although recent studies in 'micrometabolomics' and 'microproteomics' suggest that technologies are advancing in these fields7, 8. But for now, applying proteomics to a single cell is "totally impossible", says Howard Gutstein, a proteomics researcher at the University of Texas M.D. Anderson Cancer Center in Houston. Although current techniques do allow researchers to tag known proteins inside a cell, it is not possible to analyze a whole proteome or to determine differences in protein expression between individual cells, he says.
Fluidigm continues to develop chips with single-cell applications and, with a grant from California's Institute of Regenerative Medicine, is currently developing a cell-culture chip for automated differentiation of stem cells (or, in the case of induced pluripotent stem cells, dedifferentiation). The research is based on previous work in Quake's lab, and Unger predicts that the chip will be available in two years9.
"The challenge of developing a new technology that will be widely accepted for single-cell stem cell measurements is quite high", says Zandstra, especially for validation studies to prove that a candidate is indeed a stem cell. For that, functional assays remain the gold standard, such as the generation of a tetraploid mouse in the case of suspected mouse embryonic stem cells or long-term reconstitution of a mouse haematopoietic system for potential HSCs. "Ideally, what we'd like to do is ask what markers are associated with that cell so we don't have to do this ridiculous assay," he says.
A major concern
In spite of their limited application so far, technologies for examining single stem cells are generating a great deal of enthusiasm, and proponents point out that the technologies are more than just exciting, they are absolutely essential to the field. One cannot determine what pathways are directing cell specification without looking at a single cell, says McKay. "If you have a population of cells, you're lost...If you can't actually look at the given cell and ask what state is it in, then you can't conclude that you know when fate has been specified." Worthington agrees: "Each stem cell needs to be treated as an individual because they're not necessarily in the same state at every point in time. You have to look at them as individuals."
What's more, single-cell analysis may be a prerequisite in some circumstances. For therapeutic applications, stem cells will have to undergo detailed in vitro characterization before grafting. "Say I have a therapy where I need 109 cells," says Jervis. "As an engineer, I need to manufacture 109 perfect things, because if one of those cells is transformed and is going to give rise to a tumour, I now have a serious problem." He pauses. "It is a single-cell problem. I think this is one aspect with which the field hasn't come to grips. Single-cell analysis is going to be where it's at."
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Author affiliations
Megan Scudellari is a freelance writer based in Durham, North Carolina.
