It is stem cell research鈥檚 ultimate prize 鈥� stem cells made from an individual鈥檚 own cells without the need for a donated egg or embryo. Now, three research groups claim to have done just that 鈥� by reprogramming adult mouse cells into cells that are virtually indistinguishable from embryonic stem cells (ESCs). If they can repeat their success in humans, hopes are high that such cells could one day be used to regenerate healthy tissues in people with a range of illnesses.
Last year, Shinya Yamanaka and his colleagues at Kyoto University in Japan claimed to have produced ESC-like cells by exposing mouse skin cells to four chemicals only found in embryonic cells (New 杏吧原创, 11 October 2006, p 8). But while these 鈥渋nduced pluripotent stem cells鈥� (iPS cells) could develop into all types of tissues in the mouse, live animals could not be generated from them, suggesting that they were not true ESCs. 鈥淭he cells were kind of partially reprogrammed,鈥� Yamanaka says.
Now, in three papers published simultaneously this week, Yamanaka and two other groups report that by turning on expression of the same four chemicals in adult mouse cells, the cells run their differentiation process backwards, reverting to an ESC-like state (Nature, DOI:10.1038/nature05934 and DOI:10.1038/nature05944; Cell Stem Cell, DOI:10.1016/j.stem.2007.05.014). 鈥淲e have shown that cells can be generated by these four factors, that are indistinguishable from embryonic stem cells,鈥� says Konrad Hochedlinger of the Harvard Stem Cell Institute, who wrote one of the papers (watch a video of Marius Wernig 鈥� first author on the same paper 鈥� describing the cells 鈥� 3.8 MB, .wmv).
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鈥淲e have shown that cells can be generated by these four factors, that are indistinguishable from embryonic stem cells鈥�
The epigenetic patterns of the cells 鈥� signals the cell adds to DNA molecules to help orchestrate gene expression 鈥� were nearly identical to those of true ESCs, as were the overall patterns of gene expression. Moreover, Yamanaka and Rudolf Jaenisch at the Whitehead Institute at MIT in Cambridge, who wrote the third paper, proved that the cells could become functional reproductive cells and create a whole organism, the gold standard for ESCs. This time, early embryos injected with iPSCs survived to adulthood and were subsequently able to mate with females and produce their own embryos containing iPSC-derived cells. 鈥淭hat鈥檚 absolute proof that you鈥檝e got the real thing,鈥� says Robert Lanza of Advanced Cell Technology in Worcester, Massachusetts.
Stem cells derived from an adult cell have previously been created in animals through cloning 鈥� as was done to make Dolly the sheep. In this case, the genetic material of an adult cell is transferred into an egg whose own DNA has been removed. Unknown factors in the egg reprogram the adult nucleus and allow the resulting embryo to make stem cells or grow into an individual (see 鈥淓gg-free cloning鈥�). But this method often causes abnormalities in the animals and has not advanced significantly since Dolly emerged 10 years ago. 鈥淒olly was a black box,鈥� says Marius Wernig, part of the team led by Jaenisch. 鈥淗ere, we have defined conditions and you can observe what is going on.鈥�
To create the improved iPSCs, Yamanaka and the other two groups inserted genes encoding the four factors, as Yamanaka did previously, but changed the way they identified the reprogrammed cells. In his original work, Yamanaka used genetic engineering to create cells that would express an antibiotic-resistance gene only when they were also expressing a gene called Fbx15, which is active in ESCs. By growing the cells in the presence of this antibiotic, only those cells that had reprogrammed survived. But Fbx15 is not essential for maintaining an ESC-like state.
This time, all three groups chose to link the antibiotic resistance to an essential ESC gene called Nanog, which the researchers believe forces the cells to become more ESC-like. 鈥淯sing this different selection approach was critical,鈥� Hochedlinger says.
However, for all its promise, many hurdles will need to be overcome before the technique can be used in humans. 鈥淲e don鈥檛 know whether human cells can be reprogrammed at all,鈥� Wernig says. 鈥淔or sure it will be more complicated.鈥�
For one thing, the combination of factors needed to reprogram human cells may differ from those in mice, and more than four factors may be required. Yamanaka鈥檚 trials of the same four factors in human cells have so far not succeeded in making iPSCs.
Even with the factors in hand, 鈥渢he approach requires serious genetic modification of the cells,鈥� says Lanza. This would be unacceptable if cells were to be used in human therapies. To introduce the four factors into the cells, the teams use retroviruses, which incorporate each factor鈥檚 genes at random locations in the genome. This has the potential to activate tumour-causing genes. To get around this problem, Hochedlinger is trying to add the genes using a different type of virus, which remains in the cells temporarily and does not incorporate the genes for the factors into the genome. Such an approach may be feasible, since the factors are only needed at the beginning of the process.
Another concern is that one of the factors, c-myc, is encoded by a potent cancer-causing gene, so it might be reactivated down the line if left in the genome. Indeed, Yamanaka found that while most of the mice that developed from embryos containing iPSCs appeared normal, about 20 per cent developed tumours, resulting from reactivation of c-myc. 鈥淚f you were using this method to create human ESCs that could be used in therapy, there would be concerns,鈥� says Azim Surani of the Gurdon Institute in Cambridge, UK.
The process is also very inefficient, with less than one in every 1000 cells the retrovirus infects going on to express Nanog and become iPSCs. 鈥淥ne possibility is that there is a minority of cells that are more susceptible to reprogramming than others,鈥� Surani suggests. If this were true it might be possible to modify the skin cells, making them more susceptible to reprogramming.
How long it will take to solve these problems is anyone鈥檚 guess. 鈥淚 don鈥檛 know whether this will be doable in the next few years or in my lifetime,鈥� says Lanza.
Meanwhile, the approach offers a defined system for studying reprogramming. 鈥淭here is a real possibility of doing this much more systematically to get some kind of understanding of what happens to cells when they undergo differentiation,鈥� says Surani. Robin Lovell-Badge at the National Institute for Medical Research in London, agrees: 鈥淲e are starting to find what is in that black box.鈥�
(See 鈥淪tem cell therapies without tears鈥�)
Egg-free cloning
Dolly-style cloning experiments may no longer require a fresh supply of donated eggs. Kevin Eggan and colleagues at Harvard University have successfully cloned mice by inserting DNA from an adult cell into a zygote 鈥� the earliest stage embryo 鈥� with its chromosomes removed, rather than into an unfertilised egg.
If the technique can be replicated in humans, it could reduce the urgent need for donated eggs for embryonic stem cell (ESC) research. These are in very short supply. Researchers could instead use frozen early-stage embryos 鈥� including those with serious genetic defects that could not develop into viable offspring 鈥� of which many more are available.
In cloning, the chromosomes are removed from the unfertilised egg and replaced with an adult nucleus. Unspecified factors within the egg then reprogram the adult DNA to behave more like an undifferentiated embryonic cell. Researchers have already tried the same procedure in fertilised eggs, removing the two pronuclei, which contain the maternal and paternal DNA, from the egg before they fuse 鈥� but with no success.
Eggan wondered whether the reason for this failure was that the factors needed to reprogram the adult nucleus get packaged into the pronuclei, so that when they are removed, the factors go, too. His team waited for the next step in the cell cycle, when the male and female pronuclei fuse with each other 鈥� releasing the reprogramming factors into the cytoplasm 鈥� and the cell prepares to divide. His team removed these chromosomes, replacing them with those from an adult cell, and found that the resulting cells developed into mice, although they died soon after birth. He was also able to derive ESCs from these embryos.
鈥淭his confirms what many of us have suspected for a long time 鈥� that early-stage embryos can still reprogram cells,鈥� says Robert Lanza of Advanced Cell Therapeutics in Worcester, Massachusetts. It may also help in identifying more of the factors needed to reprogram adult cells, says Robin Lovell-Badge of the UK鈥檚 National Institute for Medical Research in London. 鈥淲e now have a clear idea that they are nuclear factors,鈥� he says.
However, Lanza cautions against immediately extrapolating the work to humans. His team has also managed to reprogram adult cells in mice and cows using embryos rather than eggs, but he has failed with human embryos, where even nuclear transfer into eggs has not yet worked. And as is typical with cloning, reprogramming of the adult chromosomes in these embryos seems to be incomplete: none of the mice born in Eggan鈥檚 experiment survived, and many had the same health problems as animals cloned from unfertilised eggs.
