Sunday, October 05, 2008

Induced pluripotent stem cells IV

Induced pluripotent stem cells (iPS cells) may again be judged one of the most significant scientific developments this year, and the news keeps coming. (Of course, it was near the top last year also.)

Some of our previous discussions are here, here, and here.

The ability to turn nearly any type of adult cell into the equivalent of a pluripotent stem cell seems almost too good to be true. And so far, that goal is still elusive, as a practical matter, with respect to treating diseases.

There have been at least three principal difficulties with experimental processes reported so far.

  1. The process depends on a kind of gene therapy that inserts a few desired additional genes into cellular DNA using certain types of viruses, and the process itself significantly raises the chances of affected cells becoming cancerous.
  2. One or more of the genes that are added to cellular DNA to induce pluripotency can also raise the chances of a cell becoming cancerous.
  3. Alternative methods that address the first two problems tend to be substantially less efficient, therefor slower and more expensive, for producing the desired iPS cells.

The research we'll consider here addresses the first of these problems.

Induced Pluripotent Stem Cells Generated Without Viral Integration
Pluripotent stem cells have been generated from mouse and human somatic cells by viral expression of the transcription factors Oct4, Sox2, Klf4, and c-Myc. A major limitation of this technology is the use of potentially harmful genome-integrating viruses. Here, we generate mouse induced pluripotent stem cells (iPS) from fibroblasts and liver cells by using nonintegrating adenoviruses transiently expressing Oct4, Sox2, Klf4, and c-Myc. These adenoviral iPS (adeno-iPS) cells show DNA demethylation characteristic of reprogrammed cells, express endogenous pluripotency genes, form teratomas, and contribute to multiple tissues, including the germ line, in chimeric mice. Our results provide strong evidence that insertional mutagenesis is not required for in vitro reprogramming. Adenoviral reprogramming may provide an improved method for generating and studying patient-specific stem cells and for comparing embryonic stem cells and iPS cells.

Perhaps a little more explanation would be in order. We've discussed the four transcription factors, Oct4, Sox2, Klf4, and c-Myc, in earlier articles.

The "insertional mutagenesis" mentioned here refers to the use of a type of virus that inserts some of its genes directly into the cellular DNA. Such viruses include lentiviruses and other retroviruses. HIV is an example of a lentivirus. The genetic material of this kind of virus is in the form of RNA, which must first be translated into DNA (by an enzyme called reverse transcriptase) and then integrated into the host cell DNA. The transcription factor genes are artificially added to the virus RNA so that they are copied along with everything else.

That integration step is what enables genes for the transcription factors to be inserted into host DNA. Although genes for those factors are already present, of course, in non-stem cells, they are in a form that is less readily translated into proteins than in pluripotent cells. The newly-integrated genes, however, can be easily translated, and they produce the protein transcription factors that go on to turn the cell into a pluripotent stem cell.

The drawback of this method is that the new genes can be inserted at arbitrary points of the host DNA, and this can harmfully affect other genes, which may make the cell susceptible to becoming cancerous.

What the research in this latest work has done is to add the transcription factor genes to a different kind of virus, called an adenovirus. The significant difference of an adenovirus from a retrovirus is that the genetic material of the former consists of double-stranded DNA, like the DNA of the host cell. When an adenovirus infects a cell, its DNA floats freely within the cell, and it can be translated into proteins by the same process as for the host cell DNA.

So what the new work described in this study does is to add the transcription factor genes to the adenovirus DNA, and then allow the virus to infect normal adult cells. This has the advantage of not damaging the host DNA, because it does not get integrated into it.

Naturally, this is an obvious approach to try, and it has been attempted before, but not successfully. The reason it hasn't worked before, probably, is that the adenovirus DNA is diluted every time an infected cell divides, since there may be, at most, only a couple dozen copies of the virus DNA in each infected cell. It does not get copied reliably into daughter cells, and that's a good thing on the whole. (Otherwise an infection might never go away.)

Fortunately, it turns out that simply having the adenovirus-carried transcription factor genes in a cell for a sufficiently long time can trigger further gene expression that confers pluripotency – which remains even after the virus genes are no longer present. Persistence pays.

The research was carried out using various types of mouse cells – fetal liver cells, adult hepatocytes, and fibroblasts from the mouse tail tip. The last of these can, of course, be obtained quite easily.

There is, however, a downside. The efficiency of inducing pluripotency by this method is still very low. Typically, only 0.0001% to 0.001% (1 in a million to 1 in 100,000) of cells are converted. This compares with 0.01% to 0.1% when DNA-integrating viruses are used.

The research proceeded to compare the adenovirus-induced pluripotent cells with natural pluripotent cells. The similarities were quite close:

  • Pluripotency genes of the reprogrammed cells lack methylation (a chemical modification that inhibits expression), just like the genes of natural pluripotent cells.
  • The pluripotency genes (including Oct4, Sox2, Klf4, c-Myc, and Nanog) of normal pluripotent cells are also expressed in the reprogrammed cells, even after all traces of adenovirus DNA are gone.
  • The iPS cells formed teratomas (cell masses consisting of many different cell types) when injected into adult mice.
  • When the iPS cells were injected into mouse blastocysts, which then developed into mostly normal, but "chimeric", young mice, evidence of descendants of the iPS cells turned up in many different tissue types.

The next step for research in this direction will be to find out whether the low efficiency of adenoviral reprogramming can be improved by techniques similar to those used to improve the efficiency of retroviral reprogramming.

News reports on this research:
M. Stadtfeld, M. Nagaya, J. Utikal, G. Weir, K. Hochedlinger (2008). Induced Pluripotent Stem Cells Generated Without Viral Integration Science DOI: 10.1126/science.1162494

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