Transfer of somatic nuclei into the oocyte
The problems posed by the transfer of the nucleus of somatic cells into the oocyte have been considered by four researchers, Ian Wilmut, Keith Campbell, John Clark and by Jean-Paul Renard. This method of reproductive cloning has been widely applied to mammals since the birth of the Dolly sheep in 1997 demonstrated its feasibility.
Indeed, although the reprogramming by the cytoplasm of the oocyte of nuclei coming from cells in the process of differentiation (and even differentiated) has been demonstrated in Amphibians by the experiments of Robert Briggs and Thomas King then of John Gurdon, the attempts performed to clone mammals had long been unsuccessful. Some researchers even considered that this class of vertebrates was not suitable for these experiments. In 1984, however, Willadsen successfully cloned sheep by transplanting nuclei from cells of early embryos (stage 8-16 blastomers).
However, the rate of embryonic development was only 2.5%. Cloning of a mammal from the nucleus of an adult cell was not expected until 1997. The Roslin Institute team was responsible for it. The journal Nature published an article on February 27, 1997, reporting that a clone of a sheep that had been dead for several months had been obtained. 277 nuclear transplants were required for a viable fetus to reach gestation and survive after birth. The origin of the nuclei used in this experiment was a culture of mammary gland cells taken from the original sheep and kept alive by successive subcultures.
Reproductive cloning techniques applied to mammals are an important part of the Roslin Institute’s activity and Ian Wilmut provided an overview of the prospects offered by this technology. To date, clones have been obtained in several species of mammals (sheep, cow, mouse, goat, pig, cat) while the tests carried out in the dog and the rhesus monkey have been unsuccessful. The cell types used to collect the nuclei are diverse. The survival of the clones decreases during the experiment since in vitro fertilization passing through the embryo, the fetus and then the adult.
In addition, animals that reach adulthood suffer from many anomalies. At birth, they are generally more than normal. This condition is prolonged by obesity (observed in particular in mice). Other symptoms are commonly seen such as difficulty breathing, impaired functioning of the immune system leading to reduced longevity. These anomalies appear to be epigenetic in nature. Jean-Paul Renard returns to the pathology of fetuses produced by cloning in his laboratory. The researcher’s extensive experience in cloning cattle and mice leads him to the notion that fetal mortality increases with the age of the individuals from whom the nucleus donor cells were removed.
In cloning experiments, the death of the fetuses, which is frequently observed, is accompanied by anomalies such as an enlarged placenta, that of the tongue and the head, etc. Nuclear reprogramming is the most critical phase of the process cloning. It involves modifications in histones, proteins closely associated with DNA and which participate in the control of transcriptional activity. To facilitate this critical phase, the author recommends the use of metaphase nuclei taken from cultured fibroblasts.
Cell therapy and transfer of somatic nucleus into the oocyte.
Embryos obtained by nucleus transfer from adult cells can be used, like zygotic embryos, to produce ES cells. Produced in humans from the somatic cells of a patient, they could constitute a privileged source of differentiated cells usable in cell therapy. Indeed, they would have the same genome as the patient for whom these cells are intended and would therefore not be subject to the immunological rejection triggered by heterografts.
The implementation of this technique, commonly called therapeutic cloning, is clearly distinguished from reproductive cloning since it results in the production of cell lines but in no case in the development of an individual. However, it is prohibited in many countries although it can be useful in the treatment of certain conditions such as Parkinson’s disease and for the production of cardiomyocytes.
The fact that islets of insulin-producing cells were obtained from ES cells of mice (Ron McKay) also suggests that they may be used to treat certain forms of diabetes.
Interest of ES cells obtained by somatic nucleus transfer for cell therapy in humans
John Clark summarized the state of the question and presented, from a critical point of view, the real interest that such cells could have in humans, as well as the difficulties of various kinds which accompany their use. According to this author, in the current state of our knowledge, the limiting factor in the production of cells by transfer of somatic nucleus into the oocyte (apart from ethical problems) is the number of human oocytes required to obtain the differentiated cells. necessary for the patient.
Thus, if we had 400 “good quality” oocytes, we could hope to obtain, after in vitro fertilization, a rate of 10% of development reaching the blastocyst stage, ie 40. From blastocysts we estimate 20% the chances of obtaining stem cell lines, ie only 8 lines. From these lines, a complex technology must be implemented to obtain a homogeneous culture of differentiated cells (not containing pluripotent stem cells) capable of being injected.
This is personalized medicine, which is opposed to the more or less “universal” therapeutic techniques used until now and which stands out in particular for its very high cost. According to John Clark, transplanting cells from already established lines seems more realistic today. While it is true that transplantation of heterologous cells is likely to cause immunological rejection reactions, these can be combated by immunosuppressive treatments which have been widely proven in organ transplants.
In addition, obtaining a certain degree of tolerance of the host vis-à-vis the starting ES cells does not seem unthinkable.
More specifically, in the case of type 1 diabetes for which this technique is envisaged, the destruction of the insulin-producing islets of Langherans (B cells) is the result of a disturbance of his immune system. Islet cells are destroyed by cytotoxic T cells, as would be grafted cells from a third party (allograft) or self cells infected with a virus (modified “self”).
The islet transplant with the host’s MHC (autograft) would therefore be in danger of being destroyed, as was the individual’s normal cell quota. The usefulness of such transplants is therefore likely to be zero. Their use in Parkinson’s disease may be beneficial, but there is no indication that it would be more beneficial than that of neurons from an allogenic stem cell bank.
Indeed, heterografts of embryonic mesencephalic cells practiced in humans for several years do not appear to be the subject of immunological rejection. The major utility of isogenic cells would relate to cardiomyocytes.
Are there unquestionable reasons today for producing embryonic stem cells by somatic nucleus transfer without the oocyte?
Several speakers argued in favor of the development of this technology for: – the study of variations in the toxicity and metabolism of drugs depending on the genetic background. These may be of an unsuspected magnitude – Certain diseases with a genetic component could be studied on lines obtained by this method in a thoroughly unthinkable way on affected individuals.
According to John Clark, nuclear transplantation into the human oocyte is unlikely to be a valid technology for cell therapy. It is therefore premature because it is not justified in the current state of technology to arouse, in patients, hopes for a cure based on the implementation of nuclear transfer for therapeutic purposes. However, this method, if properly supervised from an ethical point of view, should prove to be important for research.
What can we keep in mind?
Although very encouraging data emerge from the experiments carried out so far, it can be seen that much research remains to be done in particular to improve the differentiation of the cells in culture as well as the safety of the grafts which must imperatively be free of pluripotent stem cells. because of the risk of cancerous transformation they present