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Human clones and reprogrammed skin cell

Over the last few weeks, events have followed in quick succession in the field of embryonic stem cell research. Breakthrough after breakthrough has followed progress after progress. Those interested in the field of embryonic stem cell research find it difficult to study the numerous publications on this topic in detail. Non-experts will soon lose their way in the large number of publications. BIOPRO Baden-Württemberg will present the new methods and stem cell experts, regeneration biologists, medics and ethicists will explain whether they think that the new methods are useful or not.

The greatest uproar among non-scientists has recently been caused by a paper published by the team of the American Andrew J. French who succeeded in cloning a human embryo from a skin cell. The online publication in the journal Stem Cells hit the headlines in all media and was discussed more or less heatedly. However, reactions were more cautious than four years ago when the world was taken in by the South Korean clone forger Hwang.

Clear evidence

French and his colleagues removed the nuclei from female oocytes and subsequently introduced the nuclei of male skin cells. This method, which scientists refer to as somatic cell nuclear transfer (SCNT), is anything but new. It has already been used to create the sheep Dolly. However, it took years for the method to be ready for human application. It seems that five of French’s constructs developed into cloned blastocysts. This is clear evidence that a blastocyst can develop from an enucleated oocyte and the nucleus of an injected skin cell.
In cloned human embryos it is still impossible to do what is shown in the photo: the creation of human stem cell lines, which can be differentiated into specific cell types. A nerve cell could be generated from a stem cell culture. (Source: Follow the Money- The Politics of Embryonic Stem Cell Research. Russo E, PLoS Biology Vol. 3/7/2005, e234/Images: Nissim Benvenisty)
French’s team were however unable to achieve the actual goal and were unsuccessful in creating embryonic stem cell lines from the blastocysts. It is hoped that these cells, when cultured in the test tube in the presence of growth factors, will develop into different cell types, such as nerve cells, pancreatic cells or muscle cells, or even differentiate into complete organs. The plan is to then use this tissue for the treatment of degenerative diseases. This is why the entire process is often referred to as “therapeutic cloning”. If it were possible to establish the process, such as the supporters of therapeutic cloning envision, then this would prevent the recipient’s immune system from rejecting the cultivated tissue right from the start. A somatic cell of the patient and a donated oocyte would be sufficient to generate genetically compatible replacement tissue. However, this must honestly and seriously be regarded as a distant goal.

In 2008, the reality is different. The fact that it is actually possible to generate stem cells from cloned primate embryos was announced in the journal Nature in 2007. James Bryne, who works in the laboratory of Shoukhrat Mitalipov, tried his luck with SCNT in Rhesus monkeys. He also used skin cells, but in contrast to French, the team from Oregon succeeded in cultivating two stem cell lines from monkey blastocyts. According to the authors, these cell lines could be differentiated into different cell types.
In 1998, James Thomson was the first to generate stem cells from human embryos. He used what are referred to as surplus embryos. In contrast to Germany where this method is illegal, in many countries more embryos are produced for in vitro fertilization than are actually implanted into the uterus. These surplus embryos will either be discarded or – in agreement with the biological parents – used for research purposes. Embryonic stem cells are particularly interesting for research because they have a unique capacity: Under suitable conditions, embryonic stem cells can differentiate into nearly all types of somatic cells. This capacity is usually referred to as pluripotency.
How viable stem cell lines can be generated from cloned blastocysts is far from being the only problem researchers who wish to do therapeutic cloning have to deal with. It is also difficult to get hold of large enough quantities of oocytes for the experiments. British scientists are now hoping to circumvent this bottleneck with chimeras. Their officially approved plan is to create clones consisting of human nuclei and enucleated animal oocytes. In Germany, this would be as illegal as therapeutic cloning itself. In Germany, it is illegal to destroy embryos for the purpose of creating stem cell lines from blastocysts.
Figure: Derivation of patient-specific stem cells to study inherited human diseases, the role of mtDNA, epigenetic, genetic and differentiation profiles and abilities of pluripotent stem cells. Abbreviations: hESC: human embryonic stem cells; NTSC: nuclear transfer stem cells; iPS cells: induced pluripotent stem cells; MII: metaphase II; mtDNA: mitochondrial DNA. Published with the kind permission of AlphaMed Press. (Source: Cervera, R. P. und Stojkovic, M., 2008, Commentary: Somatic Cells Nuclear Transfer - Progress and Promise, Stem Cells, doi:10.1634/stemcells.2008-0025.)
Whether or not the work of Robert Lanza, which was published at the beginning of January in the online edition of Cell Stem Cell, will be able to partially resolve the problems faced, is highly controversial. Lanza is hoping to create embryonic stem cells without having to destroy embryos. His group mainly used the principle of preimplantation diagnostics, which is forbidden in Germany. The researchers always removed one cell from eight-cell human embryos which were created in the test tube. The idea behind these experiments is the following: The stem cells produced this way will be used later in a person’s life as the source for autologous tissue and organ replacements. Prerequisites for doing this will however always be that the beneficiary is actually created in the test tube rather than naturally.

Many hopes are pinned on the cryptic abbreviation iPS

Prof. Shinya Yamanaka in Heidelberg (Photo: DKFZ)
The greatest hopes – at least in Germany – are currently being pinned on the experiments involving induced pluripotent stem cells (iPS). Following the success of the Japanese Shinya Yamanaka and the German Rudolph Jaenisch, who were able to reprogramme mouse skin cells to form embryonic stem cells, Yamanaka succeeded in doing the same with human cells in November 2007. Using efficient transcription factors and efficient gene shuttles, the different work groups succeeded in reprogramming skin cells back into a near embryonic state, without having to involve embryos.

Carcinogenic potential cannot be excluded

It cannot be excluded that the cells created in this way are potentially carcinogenic. The gene shuttles, which introduce juvenating transcription factors into the genome, are retroviruses. They, along with their freight, insert into different parts of the genome where – in the worst case – they not only destroy important genetic information, but also switch on neighbouring genes that should actually remain silent. In addition, the transcription factors also have, at least partially, an undisputed carcinogenic potential.

It turns out that a dubious constituent of the juvenating cocktail, the protooncogene c-myc, does not seem to be necessary for the reprogramming, as Jaenisch was able to show a few weeks ago. Some researchers are already thinking aloud about the possibility of completely refraining from the introduction of additional transcription factor genes. This is because the information for the switches which induce the reprogramming are already available in the genome, albeit silenced in normal somatic cells. That is why some experts believe that it is possible to activate the dormant transcription factors with biochemical stimuli. But also in this case, the road to success is still a long way off.

kb – 04.02.2008
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