Induced pluripotent stem cell

Induced pluripotent stem cells ( iPS) cells are pluripotent stem cells that are created by in vitro reprogramming of non- pluripotent somatic cells. The conversion is initiated by externally stimulated expression of specific genes ( transcription factors ) in the cell of the body for which there are several techniques. iPS cells resemble stem cells in many natural properties of strongly whether today iPS cells match in all properties with natural stem cells, is an open question. Induced pluripotent stem cells have a high medical potential, because the research on them fewer ethical problems than the pulls on embryonic stem cells. In addition, specially adapted to patient iPS cells can be generated.

After 2006 in the laboratory of the first Shin'ya Yamanaka iPS cells were produced, the research on iPS cells is now one of the fastest evolving areas of biology. For the development of induced pluripotent stem cells Shin'ya Yamanaka received the 2012 Nobel Prize in Physiology or Medicine.

Production of iPS cells

Discovery

Both embryonic stem cells ( ES cells) were fused with cells of the body, as well as the cells of the first cell divisions after a somatic cell nuclear transfer are able to reprogram somatic cells to a pluripotent state. It was also before 2006 succeeded in several experiments to change by overexpression or underexpression of individual transcription factors to the cell type of somatic cells ( transdifferentiation ). Shin'ya Yamanaka presented on these foundations based on the hypothesis that genes that play a particularly important role in ES cells may also be able to restore a body cell into a pluripotent state. Together with Kazutoshi Takahashi, he carried out experiments on fibroblasts of the mouse model organism in which the expression was stimulated by central transcription factors in somatic cells by the fact that their DNA was introduced by a retrovirus into the genome ( transduction ).

Based on a total of 24 candidate genes in the experiment he was able to show that with a combination of the four genes c -Myc, Klf -4, Oct-4 and Sox -2 reprogramming of some cells in a pluripotent state is possible. It was surprising for him here is that the Nanog gene that is essential for self-renewal of stem cells, was not needed. The cells resembled natural stem cells strongly, but were not able to produce a living chimera in the blastocyst of a mouse embryo after injection. This was Yamanaka's team in mid-2007, coinciding with two other laboratories. The major improvement in this second generation of iPS cells was that was not used to obtain the successful transformed cells Fbx15 but Nanog (the proportion of successfully reprogrammed cells in a cell culture is very low, it is in parts per thousand or lower percentage ).

Human iPS cells

The end of 2007 it was possible independently to multiple teams to generate iPS cells from human somatic cells. These studies also showed that it is possible from human iPS cells gain cells of all three germ layers.

The experiment of Yu from the laboratory James Thomson had the special feature that instead of the four Yamanaka Pluripotenzgene a different combination of genes were activated: In addition to Oct4 and Sox -2 and Nanog these were Lin - 28th This showed that it is possible to dispense with c-Myc. c -Myc is a known oncogene.

Improvements of the method

After the successful reprogramming of fibroblasts has been shown that cells in different tissues from (blood, liver, brain, pancreas, etc.) reprogrammed to pluripotency .. A major hurdle on the way to clinical applications of iPS cells, however, is that in transduction by retroviruses the genome of the recipient cell is changed, which may result in cancer. Another risk is the proto-oncogene c -Myc, which - although not essential - greatly improves the efficiency of the method.

For this reason soon became sought after methods that do not permanently alter the genome of the recipient cell and thus to avoid genetically modified organisms. One approach is to use adenovirus as a vector instead of retroviruses. Another approach is to bring the genes in the form of a plasmid in the cell, so that the chromosomes of the cell are not changed. Finally, researchers succeeded in 2009 so-called protein - induced pluripotent stem cells ( piPS cells) by the introduction of recombinant proteins to produce. In this method, the cell does not produce the necessary proteins themselves by translation, as with all previous approaches. Instead, they are - in a slightly different form, so that they can pass the cell membrane - supplied to the cell from the outside. However, most of these alternative methods achieve a much lower efficiency than the stable transfection by the original four Pluripotenzgene.

Detection methods

To prove that the reprogrammed cells are truly pluripotent stem cells, a set of procedures is necessary. One can distinguish between morphological, molecular and functional evidence options.

• Morphologically: This potential iPS cells are compared under the microscope with natural ES cells. Criteria include, among other things, the shape of cells or the time between cell divisions
• Molecular: Here the patterns of transcription and the epigenetic methylation patterns of promoter regions of specific genes between iPS cells and ES cells are compared.
• Functional: The property of pluripotency is thus demonstrated that iPS cells are injected into immune- defective mice. They form spontaneously from teratomas, which contain cells from all three germ layers and thus confirm the pluripotency of the original cells. In another important test iPS cells are injected into mouse blastocysts. For functional iPS cells viable chimeras developing so. Since there is no ethical reasons in question to produce human chimeras, it is difficult to test the tendency to tumor formation of human iPS cells.

However, iPS cells can also be combined with tetraploid blastocysts. Here, the blastocysts can form only placental tissue and the embryo must originate from the iPS cells. This stringent test could now be carried out successfully with iPS cells from mice.

Mechanism

The exact mechanism leading to the pluripotency of the process is poorly understood. Because of the low efficiency of the method, it is difficult to track cells targeted during the gradual, approximately ten-day process of reprogramming. In the first generation of iPS cells, the success rate was only 0.05%. This percentage is of the same order of magnitude as the proportion of naturally occurring stem cells in a population of skin cells so that the hypothesis emerged that not terminally differentiated cells but rare natural stem cells to iPS cells.

In the following years, this suspicion could be refuted and shown that fully differentiated cells that are able to become iPS cells. In addition, the efficiency of the method based on retroviruses by the additional addition of certain chemicals was dramatically increased to about 10%. Nevertheless, it is possible that less well differentiated cells can be reprogrammed easily.

Today, most researchers assume that the process of reprogramming is stochastic in nature, in which various "barriers" epigenetic nature must be overcome. For one must the promoter regions of those genes that are essential for pluripotency, are demethylated. The acetylation of histones must be changed during the reprogramming. It is assumed that these processes are stochastic in nature, and that a part of the original cells on the path to pluripotency stuck in intermediate states (such as the original generation of iPS cells by Yamanaka, could produce no living chimeras ). There is some evidence that in principle all cells can be reprogrammed into iPS cells, even if the time in which this is done varies greatly from cell to cell.

Potential medical applications

For medical research, iPS cells are interesting because with their help can produce patient-specific cells. This may possibly in the future the problem of immune rejection, have the conventional stem cell therapy ( SCT ), are bypassed.

It is researchers already have been able to isolate iPS cells from patients with diseases such as amyotrophic lateral sclerosis or spinal muscular atrophy and can be differentiated into neurons this. Because you can often get these cells is difficult in a natural way, this method can improve the study of diseases in the laboratory.

In mice, it has been possible, by means of transplantation of iPS cells to treat sickle cell anemia and to alleviate the symptoms of Parkinson 's disease. These methods have the time but still not yet come significant risks (formation of teratomas and other tumors ), so that a clinical application with the current state of the technology in question.

According to leading researchers in the field, therefore, the therapeutic use of iPS cells is still in the distance. A use for the study of diseases and for testing potential drugs in the laboratory could, however, in the opinion Yamanaka are widely used in a few years.

Ethical considerations and critical voices

Because iPS cells arise from cells of the body, occur in their production compared to embryonic stem cells far fewer ethical problems, Therapeutic cloning or in vitro fertilization is not necessary. For proof of identity but natural ES cells are currently also indispensable in the research on iPS cells. But the research on iPS cells itself is not free of ethical problems. Benefiting from the relatively simple process of production, it is conceivable that can be recovered in the future from iPS cells or gametes with their help human clones could be generated, which raises both ethical problems.

The extremely rapid progress of research in the field of iPS cells, combined with a strong response from the public and the media and great hopes for future new therapies, also encounters critical voices. Especially by the researchers themselves, concerns were expressed that the strong competition and the foot race -like character in the field of research could harm and to lead that is released prematurely and be done too little identity tests and long-term studies on tumor formation.