西亚试剂： the sense that it is impossible to know at the beginning w

This process is unpredictable, in the sense that it is impossible to know at the beginning which cells will reprogram, and it takes them a long time. But it is predictable in some ways. “Researchers doing it in Germany, Japan and the US will all get the iPS cells about the same time and at about the same rate,” says Alexander Meissner at Harvard University in Cambridge, Massachusetts. “The one thing we know is that it's not magic, there is a mechanism. That's good news — we should be able to find it.” And yet, Meissner says, it is “almost disappointing” how little progress there is from year to year.

From the cell's point of view, it is an immense task to overcome a fully differentiated state, which is like being in biological lock-down. Take fibroblasts, for example, the connective-tissue cells that scientists often extract from skin and try to reprogram. In the long process by which they gained their identity, these cells' DNA has been stamped with 'epigenetic' markers, chemical modifications such as the addition of methyl groups or changes to the histone proteins that package up DNA. These ensure that only genes relevant for a fibroblast are expressed. It wouldn't do for a skin cell to suddenly behave like a dividing stem cell, because that can be the route to diseases such as cancer.

Scientists now have a good grip on what happens during the first 48 hours as the four Yamanaka factors, with brute force, kick cells out of this state. In embryonic stem cells, these proteins activate genes in a 'pluripotency network' that keeps cells proliferating indefinitely. But the factors act differently when shoved into a differentiated cell such as a fibroblast. When cell biologist Ken Zaret at the University of Pennsylvania in Philadelphia mapped the location of these factors during the first two days of reprogramming in human fibroblasts, he found that they were “physically blocked” from reaching their usual target genes by the conformation of the chromosomes8.

Instead, the proteins head for accessible areas of the chromosomes. Sometimes, they activate genes that force the cell to commit suicide; in others, they bind to distant control regions called enhancers that encourage the activation of genes known to be involved in the reprogramming process. Rudolf Jaenisch, a stem-cell scientist at the Massachusetts Institute of Technology in Cambridge, has labelled this widespread binding of the Yamanaka factors as “promiscuous”9.

Other studies have illuminated the sweeping changes that take place on chromosomes during this early phase. In a study published in 2011, Meissner's group showed that a type of histone modification that boosts gene expression, called H3K4me2, changes at more than 1,000 positions in the genome of these cells: it was added at many sites on pluripotency genes, and dropped from sites where genes specific for fibroblasts reside10. At the same time, the cells look and behave differently: they compact and move around less.

“Our early thought was that the factors create complete chaos,” says Meissner. “But this first step is predictable and consistent across all cell types.” Now he can almost foretell for a given cell type “which sites might become open to active transcription, which might be modified, and which will stay silent”, he says. “That part you can predict. But that doesn't answer the question of what happens next.”

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The week-long lag that follows flummoxes scientists. The cells soldier on, and some express new genes, but not in a predictable or comprehensible way. Even the H3K4me2 modifications mapped by Meissner do not seem to boost gene expression until much later in the process. “Most cells reach a partially reprogrammed state. Some get beyond that, and we're not sure why,” says Meissner. “That is the black box.” If a cell starts to pump out Sox-2 protein, however, that is a really good sign that it is progressing. “Once Sox-2 comes on, everything falls in line,” says Jaenisch, who studied the activity of nearly 50 genes in individual cells as they went through reprogramming11. Within a few days, the production of this and other transcription factors necessary for pluripotency all ramp up.

But why does all this take so long, and why is it so rare? “We don't understand why it can't be faster,” says Woltjen. He suggests that a cell might need to go through several divisions, each taking at least half a day, to reshape its epigenetic state. “Perhaps that's one limiting factor,” he says.

Yamanaka offers several possible explanations for the low conversion rate. One is that the starting cell population is a rainbow of cell types. The chunk of tissue used to derive fibroblasts, for example, probably contained a mix of subtly different cell types; even those that are fibroblasts will differ slightly in the blend of proteins and other molecules they contain. Furthermore, cells growing in culture are constantly shuttling back and forth between different states. This means that the introduced reprogramming factors will affect each cell differently. “What works for one subset of the population will not work for others,” Yamanaka says. Minor differences in cell culture and the relationship with neighbouring cells also make it difficult to control all the variables and command the cells like an obedient army, he adds. “A perfect implementation is impossible.”

Researchers are now trying to classify some of the cell types that come out of the black box, and are tinkering with reprogramming techniques to see if they can pin down how and where they diverge. Woltjen, for example, has shown that the ratio of the different reprogramming factors affects the type of cells produced. One set of conditions has a high success rate, but the resulting cells end up in a partially reprogrammed, unstable state; another has a low efficiency but produces mainly high-quality iPS cells.

Project Grandiose has also supported the idea that variability in the reprogramming process is producing fundamentally different cells. The project, launched in 2010 by some 30 senior scientists at 8 research institutes, was motivated by Nagy's desire to open up the black box. “I wanted to find out what was in it,” he says. After triggering reprogramming with the Yamanaka factors, the team collected 100 million cells per day for a month, and then regularly analysed their production of protein and RNA, their changing methylation state and more. The methylation analyses alone produced so much data that collaborators resorted to sharing it on terabyte hard drives that they FedEx-ed around the world. The size of the undertaking also inspired the project's title, Nagy says. “The name just came out of my head when I was considering how much data was being collected,” he says.

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