Last update: Mai 2014
Contact: Theresia Volhard
I. Medical and scientific aspects
The sexual reproduction for all living organisms, except for bacteria, takes place especially through the formation and recombination of germ cells (sperm cells and egg cells). A new genome originates from the composition between paternal and maternal genotypes. The term “cloning” is in contrast a form of asexual or vegetative multiplying, in which the genome of the respective organism is duplicated. In the process of cloning, there is no reorganization (recombination) of genes; rather a nearly or even completely identical genetic “copy” of the original organism arises. Beside sexual reproduction, cloning is a usual way of reproduction for many invertebrates and for most plants. The natural occurrence of identical twins (monozygotic twinning) in humans is also considered a form of identical multiple formation, yet it occurs only in the context of sexual reproduction.
In the laboratory, organisms can be cloned in different ways. One way is the division of already existing embryos, meaning through embryo splitting. Another method is the creation of an embryo by means of cell nuclear transfer. A further procedure is known from the generation of so-called transgenic mice, namely the tetraploid complementation assay (see module tetraploid complementation assay). Beside these variations in the cloning technique, different purposes of cloning should be differentiated, namely between research and therapeutic cloning on the one side, and reproductive cloning on the other side. The target of all cloning techniques is the production of a genetically identical duplicate: a DNA fragment or molecule, a cell, a tissue or in the case of reproductive cloning, a complete organism.
The object of this focal point is cloning for research and therapeutic purposes. Reproductive cloning, however, is only considered insofar as the techniques are identical to a certain degree.
To avoid possible misunderstandings, the term therapeutic cloning, which dominates the public debate, will be substituted and complemented with the term “research cloning” in the following text. A major part of the current research does not aim at concrete therapeutic purposes; rather it mainly belongs to the field of basic research. This basic research can and should lead to the development of new therapies on the long run, but is also used above all to achieve a basic understanding of the scientific relevant processes.
What is research cloning or therapeutic cloning?
Different methods are summarized under the term “research cloning” or “therapeutic cloning” such as cell nuclear transfer, the reprogramming of differentiated cells and embryo splitting.
Using cell nuclear transfer (CNT) (see module Cell Nuclear Transfer) or somatic cell nuclear transfer (SCNT), the nucleus of any somatic cell is transferred into an egg cell whose nucleus has been removed. The nucleus can be isolated from practically any adult somatic cell of a donor. The egg cell gained by means of puncture from the ovaries of a donor after a special hormonal treatment is then enucleated. The enucleation takes place using a micro pipette which sucks off the nucleus of the egg cell and substitutes it with the nucleus extracted from the somatic cell. The transfer of the new nucleus takes place through injecting the nucleus into the cytoplasm of the egg cell. The egg cell sends out impulses whose mode of action is so far unknown. These impulses bring about a reprogramming of the cell nucleus which causes the nucleus to lose its specialization. This way the nucleus will be restored from its already differentiated state back to the state which enables the development of the embryo.
Considering the genetic material contained in the nucleus, the cloned embryo is genetically identical with the donated nucleus. The Mitochondria, i.e. the cell components which serve the production of energy inside the cell, are derived from the egg cell. The clone developed as a result of the cell nuclear transfer is genetically almost completely identical with the donor of the transferred nucleus. A complete identity is only accomplished, when the donor of the nucleus and the donor of egg cell are genetically identical.
The method of cell nuclear transfer gained recognition through the results of the team of Ian Wilmut's research team. The team succeeded in 1997 for the first time in producing and fully developing a mammal-embryo by means of transferring the nucleus of an adult somatic cell into an enucleated egg cell. The cloned sheep Dolly stands for the success of the research, yet also for the possibility of using the cloning technique for reproductive purposes which are ethically extremely controversial.
In addition, one procedure has been known for many years now by means of which human somatic cells can be successfully reprogrammed so that they exhibit significant characteristics of embryonic stem cells. Such cells are called induced pluripotent stem cells (also known as iPS cells). Since the iPS cells are also genetically identical with the donor’s cells, this technique offers a less problematic alternative to therapeutic clones in ethical and legal terms. It is debatable whether iPS cells can evolve into totipotent cells under the condition of cell culture. However, the successful establishment of pluripotency from iPS cells of mice with the aid of the procedure of tetraploid complementation assay triggers discussion here. Considering these results, it is doubtful whether the procedure, with respect to its ethical permissibility, is brought more closely to research cloning as well as research on donated and artificially generated embryos.
With regard to the technical aspect, research cloning and cloning for reproductive purposes are not fundamentally different. However, it is decisive that the embryo in the case of research cloning is not implanted into the uterus in order to be born. Rather it gets destroyed in an early stage of the embryonic development (the blastocyst stage) (see module blastocyst stage) so that embryonic stem cells can be extracted and differentiated in vitro into specific cell types. It is not inappropriate to describe the approach as “therapeutic cloning”, since there is the hope that the cells which have become available can at the end be transferred again to the donor organism for therapeutic purposes. Currently, cloning is, however, mostly conducted for research purposes, if it’s legally permitted at all.
What is the purpose of research cloning or therapeutic cloning?
A primary target of research or therapeutic cloning is the production of embryonic cells (ES-cells). These cells are of interest for the researchers because they can, under appropriate circumstances, be developed into almost all different kinds of somatic cells. This ability is described as pluripotency (see module Pluripotency and Totipotency). It is controversial, whether the stem cells produced by means of cloning can become totipotent cells under cell culture conditions. An experimental evidence of totipotency (see module Pluripotency and Totipotency) of embryonic stem cells is prohibited for moral reasons, since it would be necessary to let a complete organism grow to maturity.
A long-term aim of research cloning is the production of autologous stem cells for therapeutic purposes. These are stem cells, whose genetic features are to a large extent identical with the genetic features of the patient to be treated. In this way it would be guaranteed that the cell or the tissue incorporated in the organism for therapeutic purposes is highly compatible with the immune system. Hence a number of complications which occur during the application of heterologous transplants (such as the case in organ donation) would be avoided. In particular it is hoped, in the case of research success, to be able to provide a transplantation medicine by means of the cloning technique by virtue of which the as of today still necessary long-term administration of immunosuppressivant would be unnecessary or at least diminished and the additional current scarcity of transplants would be relieved. However, in November 2014 researchers of the University Medical Center Hamburg-Eppendorf published in the journal Cell Stem Cell a study on mouse model (see module SCNT-Derived ESCs with Mismatched Mitochondria Trigger an Immune Response in Allogeneic Hosts) in which a rejection reaction occurred after the transplantation of SCNT-ES Cells. The reason for this are the mitochondrial differences between the transplanted cells and the ones of the receiver. Since mice show a comparably low variability in mitochondria, immune reactions are possibly to be expected in humans as well. According to opinion of researchers, the SCNT still represents, however, a promising path to new therapies when the rejection reaction is solved. However, possible risks of the therapeutic application of cloned stem cells are indicated; in particular, that stem cell therapies can induce a tumor growth. A point will have to be clarified accordingly is whether and how the emergence of tumors can be prevented.
Also the iPS cells which are genetically identical with the donor’s cells are regarded as another favorite for the mentioned research purposes. However, the procedure is currently associated with risks which must first be eliminated before implementing it within a therapeutic framework. Moreover, it has been shown that there are greater differences after all between iPS cells and pluripotent ES cells than initially anticipated. A therapy using tissue cells which are obtained from reprogrammed cells is therefore not possible at the moment. More details are available under the focal point “Stem Cell Research”.
Status of Research
Experimentations in the field of research cloning took place for a long time exclusively in the shape of animal experiments. In 2000, Munise et al. reported for the first time about the successful cultivation of pluripotent embryonic stem cells in mice. For this purpose, they injected the genetic material of mice somatic cells into the enucleated murine egg cells and let the resulted clone grow to maturity until the stage of blastocyst. The embryonic stem cells extracted from the blastocyst were further cultivated in the petri dish and differentiated into nerve and muscle cells. These stem cells were subsequently marked and injected in mice embryos and in already full-grown mice. It has been proved that the cloned stem cells in the embryo of the mouse (see module Stem Cells in Mouse Embryo) contributed to the development of brain, liver, lung, kidney and other organs and that they can also develop to build different tissue types inside mature mice.
The successful extraction of stem cells from previously cloned primate embryos (see module cloned primate embryos) was described for the first time at the end of 2007. Cell nuclei were transferred from skin cells of rhesus monkeys via cell nucleus transfer and inserted into enucleated egg cells. These stem cells developed into blastocysts, from which stem cells that were proven to be to a larger extent genetically identical with the original donor cells were be obtained. In all tested aspects, the cells corresponded to conventional embryonic stem cells and, according to the research group led by Shoukhrat Mitalipov, differentiated into cardiac muscle cells and nerve cells.
At the beginning of 2008 Tabar et al. published the results of a therapeutic experimentation on stem cells from a cloned embryo for the treatment of Morbus Parkinson in the mouse model (see module Cloned Parkinsonian Mice Embryos). Embryos were cloned from the skin cells of mice with Parkinson's disease, of which stem cells were be extracted again and developed into specific nerve cells. These nerve cells were injected in the diseased donor mice, which showed no immune response, but rather a significant relief of the disease symptoms. It is uncertain whether these results obtained from animal experiments can be transferred to humans as well.
Likewise, an American research group led by Andrew French published at the beginning of 2008 for the first time the results of the successful cloning of human embryos (see module Human Cloning). Thereby the nucleus of a human adult skin cell was removed and transferred into an enucleated egg cell. The researchers used 29 egg cell from three donors aged 20 to 24 for the experiment. The egg cells were donated by women, who donated them voluntarily when the egg cells became redundant after an IVF treatment. Five of the egg cells which were filled with the foreign genetic material developed blastocysts. This development was then interrupted by the scientists. The blastocysts prove the successful cloning which is represented in the genetic identity between the stem cell line and the donor cell.
In May 2013 the research group led by Masahito Tachibana and Shoukhrat Mitalipov extracted for the first time human embryonic stem cells from cloned embryos. The scientists had also transferred the nucleus of a human adult skin cell into a donor enucleated egg cell. By means of a systematically enhanced method, only a few egg cells were used for the study, and hence an early death of the embryo was prevented. The embryos were destroyed after a few cell divisions to extract the stem cells from them.
In April 2014 Robert Lanza from the biotechnology company ACT and Dong Ryul Lee from the Stem Cell Institute in Seoul issued (see module Cloning Stem Cells Using Adult Cells from Elderly Humans) the successful establishment of stem cell lines which they extracted from the skin cells of two adults, a 35-year-old man and a 75-year-old man, using a cloning method. It was thus possible to show, compared to the previous year, that stem cells can also be extracted using cell material which already display numerous genetic and biochemical alterations as well as suspected damages to the DNA. The aim of this therapeutic cloning is to produce a genetically identical replacement tissue from the somatic cells of the patients which does not get rejected.
To avoid the problems related to the use of human egg cells – the procedure of the egg cell retrieval (see module Egg cell retrieval) is risky in itself and furthermore associated with the administration of high doses of hormones– alternative egg cell resources are being searched. At the beginning of 2008 a British research team led by the stem cell scientist Lyle Armstrong produced embryos from a human genetic material, derived from skin cells, and egg cells, derived from cows. The embryos produced in this way were destroyed after three days. The target of the experiment, which was criticized as the creation of chimera (see module Creation of Chimera) and hybrids, is to determine, whether it is possible to use animal egg cells instead of human ones for the production of stem cells, which possibly could be used therapeutically after all.