Soon it will be clear that this year old manuscript by Kazutoshi Takahashi and Shinya Yamanaka will be the basis of a future Nobel Prize.
The paper is how to transform any cell into a stem cell. In the paper the authors took an “I’m so smart” approach to the whole problem. These smart guess approaches rarely work, but it’s always worth trying. This paper demonstrates why.
So let’s get back to the problem. Reprogramming cells so that they can become totipotent. How to proceed? From the paper:
We selected 24 genes as candidates for factors that induce pluripotency in somatic cells, based on our hypothesis that such factors also play pivotal roles in the maintenance of [embryonic stem] cell identity.
So here is a list of 24 genes that when activated COULD promote the reprogramming of cells so that they can acquire stem cell like properties. Then what? The researchers inserted the genes into vectors and then threw the mix of all 24 DNA constructs onto fibroblasts (mouse embryonic fibroblasts) that had an drug resistance marker controlled by a stem cell specific promoter. The idea is that if the MEFs became stemcells, they will turn on stemcell specific promoters and thus acquire resistance to the drug G418. All you have to do to find these “stemcells” is grow the cells in the presence of the drug and see whether any cells survive.
Sure enough they could find such cells. Wow! And not only that but these resistant cells formed colonies and according to the authors each colony
exhibited morphology similar to ES cells, including a round shape, large nucleoli, and scant cytoplasm.
The next thing to do is cut down the number of genes, after all 24 is quite a huge number.
And so they did … to four genes, each of which encodes a transcription factor.
So what are the identities of these genes?
Oct3/4, Klf4, Sox2 and c-Myc.
The authors wanted to then analyze cells that had been converted by the introduction of these four factors, now called iPS cells for induced pluripotent stem cells. They looked at overall gene expression in iPS vs embryonic stem cells, the activation of key stemcell markers in iPS cells and the ability of these cells to differentiate into other cell types. As far as the authors could tell, these cells looked like stemcells. From the paper:
We examined the pluripotency of iPS cells by teratoma formation (Figure 5A; Table S6 and Figure S3). We obtained tumors with 5 iPS-MEF10 clones, 3 iPS-MEF4 clones, 1 iPS-MEF4wt clone, and 6 iPS-MEF3 clones after subcutaneous injection into nude mice. Histological examination revealed that 2 iPS-MEF10 clones (3 and 6), 2 iPS-MEF4 clones (2 and 7), and the iPS-MEF4wt-4 clone differentiated into all three germ layers, including neural tissues, cartilage, and columnar epithelium. iPS-MEF10-6 could give rise to all three germ layers even after 30 passages (Table S6 and Figure S3). We confirmed differentiation into neural and muscle tissues by immunostaining (Figure 5B) and RT-PCR (Figure S4).
So there you go. A recipe for making stem cells.
But the more I think of it the more I’m surprised All it took to wipe the slate clean was to express four genes.
The only questions left – is it really true? Is it so simple?