As promised, I’m going through the three papers from last week about the re-programming of adult cells into an embryonic-like phenotype. Since it is three papers I’ll go through first what’s common to all three, and then what each group did special.
First of all, let’s summarize the method one more time.
All of these papers are based on the “rational identification” of 4 critical transcription factors by Yamanaka in 2006. What they did was take 20 proteins that drive the expression of other genes that were known to be in embryonic stem cells, and added them to adult cells to see which were critical for the embryonic stem cell phenotype. They screened which cells had been reprogrammed by using a control region upstream of the Fbx15 gene to drive a drug-resistance marker in the cells. Only cells which turned on the Fbx15 gene – a gene expressed in embryonic stem cells – would survive subsequent selection with the drug. Since many cells were infected and only a few transformed into the embryonic-like cells, the authors needed a way to rapidly determine which ones had done this.
They then subtracted factors one-by-one until they identified just 4 factors which seemed critical for this transformation of adult cells into ES cells. These four were Oct4, Sox2, c-Myc, and Klf4, and it was a surprise because only four transcription factors were needed to reprogram the cells back into being pluripotent, that is, capable of making many lineages of cells.
The main problems were that: (1) gene-array analysis (comparing the levels of expression of tens of thousands of genes) showed that while they were similar to embryonic stem cells, there were significant differences (2) there were problems in reprogramming the chromatin – the scaffolding molecule that binds DNA that also is critical to epigenetic regulation of gene expression – and other post-translational modifications of DNA suggesting the cells were somewhere in-between adult fibroblasts and embryonic stem cells, (3) the method used to deliver the genes – a retrovirus – creates problems for translation into therapy as it causes these genes to insert randomly into the genome, (4) the promoters used to express the genes are always on – or constitutive – and these genes can have negative consequences (cancer) when expressed inappropriately.
These new papers have replicated and expanded upon Yamanaka’s original work.
Maherali et. al. Cell Stem Cell 
The first paper I’ll discuss – from Kathrin Plath and Konrad Hochedlinger (from UCLA and Harvard respectively) was published simultaneously in Cell Stem Cell in the journal’s inaugural issue. The first major difference is these researchers, instead of using the Fbx15 gene to identify the cells, used a gene called Nanog – which is also required for the ES-cell phenotype – to drive the selection gene and the green fluorescent protein (GFP) to make it easier to visualize which cells successfully converted.
The cells from this group additionally were able to grow without a feeder-layer – a good sign for those who don’t want their custom ES cells grown in the presence of xenobiotics – and also were capable of reprogramming the ES-like phenotype onto other somatic cells – another good control to show they had the correct phenotype. They also did more testing to show that the transformation isn’t immediate. After adding the factors you had to wait a good while – about a week, for the changes to occur. This is a very long time for a cell culture experiment if you ask me, and just goes to show that the process of re-arranging the chromatin in the cells must be a gradual process.
These authors also did the proof-of-principle experiment of controlling one of the genes – Oct4 – with a tetracycline-inducible promoter which would in the future allow researchers to turn these genes off when they are no longer needed (potentially solving problem 4). These are promoters which are only active in the presence of a drug like tetracycline or doxycycline. They also can be made to work with other chemicals like tamoxifen.
Also, for those who want to know how to test pluripotency – there are a few ways. You can remove the anti-differentiation factor, LIF, and then just watch them differentiate in the dish. Then there are in vivo methods. The easiest is to inject the cells into an immune-deficient or “nude” mouse and wait for the cells to make a characteristic tumor – the teratoma – a benign tumor containing cells from the 3 primordial germ layers. The second, which is more rigorous, is to inject the cells into a mouse blastocyst and then implant the blastocyst into a female. The resulting mice will be “chimeras” or mixtures of the two cell types in the blastocyst. The resulting mice look like these ones from their last figure:
The chimera is the top one, and the lighter-colored hair is from the injected ES-cells which have genes for a brown or agouti coat. There’s a third method we’ll get into with the last paper.
Finally, for some reason not fully clear to me, their methods were a slight improvement on the original as their cells had a DNA methylation pattern closer to that of real embryonic stem cells – as well as proper demethylation of both X-chromosomes (ES cells should not have an inactivated X chromosome – if the cells are female both X chromosomes should be active until differentiation) – possibly bypassing problem 2. This might have been because the Nanog locus was better for selection than Fbx15 – the following paper’s results support this – or it might have just been something simple like how they waited 7 days rather than 3 for the cells to transform. Time will tell.
Okita et al. Nature 
This second paper, is from Yamanaka’s group at Kyoto university – the one that got this method up-and-going, uses similar methods to his original paper – but this time they demonstrate “germ-line transmission”. That is, they are able to use their technique to make embryonic stem cells from adult cells, they then make mice out of them using the chimera technique described above, and in a certain portion of these mice the cells contributed to the germ-line – sperm or egg cells – allowing them to create viable offspring from breeding. It should also be noted that this paper is an excellent example of just beautiful data-presentation. Everything is very clear and the data is very crisp and pretty – it’s more important than one would think.
Now, the first thing take away from this paper are that by switching to selection for Nanog from Fbx15 they were able to make their cells fully pluripotent and capable of making real live mice – which they failed to do in their first paper. However, they also waited 7 days after retroviral induction – so it’s hard to be sure it still isn’t a timing issue.
Here’s a picture of their mice after 2 generations – and an example of great presentation of data:
The offspring of the chimeras are ligher-colored – the light coat shows the trait has been inherited from the chimeras and passed to subsequent generations. This time the coat is uniform, because rather than being a mixture of engineered and wild-type cells, these animals were generated from a cross of the chimeric animals with normal mice – showing the cells can result in fertile offspring.
The second main thing to take from this paper is that the mice generated from this process developed tumors about 20% of the time, and the authors traced the origin to the myc oncogene (one of the critical 4 factors is a notorious oncogene). This is a major limitation of therapeutic potential. For whatever reason the other factors are silenced during development despite being transgenes – while the myc gene was refractory to silencing and this resulted in a variety of cancers in the animals. It should also be noted that they showed the cells generated with Nanog rather than Fbx15 selection were capable of being passaged longer in culture in the undifferentiated state – another excellent outcome.
Wernig et al., Nature 
The second paper from Nature is from Rudolph Jaenisch’s group at Whitehead/MIT. The main thing to take away from this paper – which also selected with Nanog and created germline chimeras much the same as the first two – is that they did an even more rigorous test of the cells pluripotency, and actually demonstrated what should be called totipotency – the ability to make a complete viable embryo and every type of cell in it.
The previous chimeric assays relied on injected the reprogrammed ES cells into a mouse blastocyst – which also contains wild-type ES cells in the inner cell mass. So the resulting embryos are a mixture of wild-type and engineered cells. It’s possible that the wild-type cells can compensate for defects in the engineered cells, and it would difficult to detect. So Jaenisch’s group went to the next level and used a technique called tetraploid rescue – it’s complicated – that allows them to generate embryos that are 100% made from the engineered cells. This shows, with a high degree of certainty, that the re-programmed cells can be totipotent.
Hopefully this hasn’t been too technical, if there is something that can be clarified, please comment away. The main things you should take away though are:
- In mice scientists have been able to reprogram adult/differentiated cells back into embryonic stem cell-like cells.
- These cells can make any cell type – they are totipotent.
- These cells can be passaged (probably) indefinitely
- There are still major problems – the use of retroviruses (random insertion into the genome), the potential for cancer from failure of one of the oncogenes to be silenced, and the need to adapt this to human cells.
Still this is a major success and improvement from last year’s results using Fbx15 which seemed to create a adult/embryonic hybrid that wasn’t fully totipotent. With time the system should be able to be adapted to human cells, and if we’re fortunate we’ll be able to eliminate the other technical difficulties that the proof-of-principle experiments were subject to.
1. Nimet Maherali, Rupa Sridharan, Wei Xie, Jochen Utikal, Sarah Eminli, Katrin Arnold, Matthias Stadtfeld, Robin Yachechko, Jason Tchieu, Rudolf Jaenisch, Kathrin Plath, and Konrad Hochedlinger. Directly Reprogrammed Fibroblasts Show Global Epigenetic Remodeling and Widespread Tissue Contribution. Cell Stem Cell, Vol 1, 55-70, 07 June 2007
2. Okita,Keisuke; Ichisaka,Tomoko; Yamanaka,Shinya. Generation of germline-competent induced pluripotent stem cells Nature. Published online 6 June 2007
3. Wernig,Marius; Meissner,Alexander; ; Foreman,Ruth; Brambrink,Tobias; Ku,Manching; Hochedlinger,Konrad; Bernstein,Bradley E.; Jaenisch,Rudolf In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state Nature. Published online 6 June 2007.