Induced pluripotent stem cell

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Induced pluripotent stem cells ( iPS cells ) are pluripotent stem cells that have been created by artificial reprogramming of non-pluripotent somatic cells . The conversion is triggered by the externally stimulated expression of special genes ( transcription factors ) in the body cell, for which various techniques exist. iPS cells are very similar in many properties to natural stem cells. Whether today's iPS cells match natural stem cells in all of their properties is an unanswered question. Induced pluripotent stem cells have a high medical potential, as research on them poses fewer ethical problems than research on embryonic stem cells . In addition, iPS cells specially adapted to patients can be generated.

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

Production of iPS cells

discovery

Scheme for generating iPS cells:
1) Formation of a cell culture of somatic cells
2) Introduction of the pluripotency genes into the cells by means of a retrovirus vector. Cells expressing the exogenous genes are shown in red
3) Harvesting and culturing the cells using feeder cells (gray)
4) A small part of the cells (red) become iPS cells.

Both embryonic stem cells (ES cells) that have been fused with body cells and the cells of the first cell divisions after a somatic cell nucleus transfer are able to reprogram body cells to a pluripotent state. In addition, several experiments before 2006 succeeded in changing the cell type of somatic cells ( transdifferentiation ) through overexpression or underexpression of individual transcription factors . Based on these foundations, Shin'ya Yamanaka hypothesized that genes that play a particularly important role in ES cells could also be able to restore a body cell to a pluripotent state. Together with Kazutoshi Takahashi , he carried out experiments on fibroblasts of the model organism mouse , in which the expression of central transcription factors in body cells was stimulated by the fact that their DNA was introduced into the genome by a retrovirus ( transduction ).

Starting from a total of 24 candidate genes, he was able to show in an experiment that a combination of the four genes c-Myc , Klf-4 , Oct-4 and Sox-2 can reprogram some cells into a pluripotent state. He was surprised that the Nanog gene, which is essential for stem cells to self-renew, was not needed. The cells closely resembled natural stem cells, but were unable to create a living chimera after injection into the blastocyst of a mouse embryo . Yamanaka's team succeeded in doing this in mid-2007, at the same time as two other laboratories. The decisive improvement in this second generation of iPS cells was that not Fbx15 but Nanog was used to preserve the successfully converted cells (the proportion of successfully reprogrammed cells in a cell culture is very low, it is in the per mille or lower percentage range) . Induced pluripotent stem cells have a partially different phenotype to embryonic stem cells. The conversion to induced pluripotent stem cells is accompanied, among other things, by removal of the trimethylation of lysine in histone H3 at position 27 (H3K27me3).

Human iPS cells

At the end of 2007, several teams independently succeeded in generating iPS cells from human body cells. These studies also showed that cells from all three cotyledons can be obtained from human iPS cells .

The specialty of Yu's experiment from James Thomson's laboratory was that instead of Yamanaka's four pluripotency genes, a different combination of genes was activated: Besides Oct4 and Sox-2 , these were Nanog and Lin-28 . This showed that it is possible to do without c-Myc . c-Myc is a well-known proto oncogene .

Method improvements

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

For this reason, methods were soon sought that would not permanently change the genome of the recipient cell and thus avoid genetically modified organisms . One approach is to use adenoviruses as a vector rather than retroviruses. Another approach is to bring the genes into the cell in the form of a plasmid so that the cell's chromosomes are not changed. Finally, in 2009 researchers succeeded in creating so-called protein-induced pluripotent stem cells (piPS cells) by introducing recombinant proteins . With this method, the cell does not produce the necessary proteins itself through translation as in all previous approaches. Instead, they are supplied to the cell from the outside - in a slightly modified form so that they can pass through the cell membrane . Most of these alternative methods, however, achieve a much lower efficiency than the stable transfection by the original four pluripotency genes.

The generation of induced pluripotent stem cells was named method of the year 2009 by the journal Nature Methods .

Detection methods

A number of procedures are necessary to prove that the reprogrammed cells are really pluripotent stem cells. A distinction can be made between morphological, molecular and functional detection options.

  • Morphological: Here, potential iPS cells are compared with natural ES cells under the microscope. Criteria include the shape of the cells or the time between two cell divisions
  • Molecular: Here, the patterns of transcription and the epigenetic methylation pattern of promoter regions of special genes between iPS cells and ES cells are compared.
  • Functional: The property of pluripotency is demonstrated by injecting iPS cells into immunodeficient mice. These spontaneously develop teratomas , which contain cells from all three germ layers and thus demonstrate the pluripotency of the original cells. In another important test, iPS cells are injected into mouse blastocysts . In this way, viable chimeras develop from functional iPS cells . Since it is out of the question to generate human chimeras for ethical reasons, it is difficult to test the tendency to tumor formation of human iPS cells.

iPS cells can also be combined with tetraploid blastocysts . The blastocysts can only develop placental tissue and the embryo must come from the iPS cells. This more stringent test could now be carried out successfully with iPS cells from mice.

mechanism

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

In the following years this suspicion could be refuted and it could be shown that completely differentiated cells are able to become iPS cells. In addition, the efficiency of the retrovirus-based method has been increased dramatically to around 10% by adding certain chemicals. Nevertheless, it is possible that less well differentiated cells can be reprogrammed more easily.

Today most researchers suspect that the process of reprogramming is stochastic in nature, in which various “barriers” of an epigenetic nature have to be overcome. On the one hand, the promoter regions of those genes that are essential for pluripotency must be demethylated . The acetylation of the histones must also be changed during the reprogramming. It is believed that these processes are stochastic in nature and that some of the original cells get stuck in intermediate states on the way to pluripotency (such as the original generation of Yamanaka's iPS cells, which could not create living chimeras). There is some evidence that, in principle, all cells can be reprogrammed into iPS cells, even if the time in which this takes place varies greatly from cell to cell.

Potential medical uses

IPS cells are interesting for medical research because they can be used to produce patient-specific cells. In this way, the problem of immune rejection that conventional stem cell therapies ( stem cell transplantation ) has, may be avoided in the future.

Researchers have already succeeded in isolating iPS cells from patients with diseases such as amyotrophic lateral sclerosis or spinal muscular atrophy and having them differentiate into neurons . Since these cells are often difficult to obtain naturally, this technique can improve the study of diseases in the laboratory.

In mice, it has been possible to treat sickle cell anemia by transplanting iPS cells and to relieve the symptoms of Parkinson's disease . At the moment, however, these methods still have considerable risks (formation of teratomas and other tumors), so that clinical application with the current state of technology is not yet an option.

According to leading researchers in the field, the therapeutic application of iPS cells is still a long way off. However, Yamanaka believes that it could be used to study diseases and test potential drugs in the laboratory within a few years.

Ethical points of view and critical voices

Since iPS cells arise from somatic cells, there are far fewer ethical problems in their production compared to embryonic stem cells; therapeutic cloning or in vitro fertilization are not necessary. For proof of identity , natural ES cells are currently indispensable for research on iPS cells. But research on iPS cells themselves is not free from ethical problems either. Favored by the relatively simple manufacturing process, it is conceivable that gametes could be obtained from iPS cells in the future , or that human clones could be generated with their help , both of which raise 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 high hopes for new forms of therapy in the future, is also met with critical voices. Concerns were expressed by the researchers themselves that the strong competition and the race-like character in the field of research could be harmful and lead to publications being published too quickly and too few identity tests and long-term investigations for tumor formation being carried out.

In 2014, a scandal surrounding two experimental publications in Nature on STAP cells , a subset of iPS cells which, according to the authors, should have acquired pluripotency through external stimuli, led to the articles being withdrawn because the results turned out to be fabricated.

literature

Individual evidence

  1. ↑ Press release on the Nobel Prize in Medicine 2012 , The Nobel Assembly at Karolinska Institutet, October 8, 2012, accessed on October 8, 2012
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  29. ↑ Interview with Rudolf Jaenisch
  30. ↑ Interview with Konrad Hochedlinger
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  33. ↑ after Baker (2009)
  34. ↑ Archive link ( Memento of the original from June 12, 2009 in the Internet Archive ) Info: The archive link was inserted automatically and has not yet been checked. Please check the original and archive link according to the instructions and then remove this notice.  @1@ 2Template: Webachiv / IABot / blogs.nature.com
  35. ↑ David Cyranoski: Papers on 'stress-induced' stem cells are retracted. Nature News, July 2, 2014 doi : 10.1038 / nature.2014.15501