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A team of scientists, for the first time, published research in Cell describing how they used somatic cell nuclear transfer (SCNT) to produce human embryonic stem cells. SCNT is a technique which generates embryos that are almost an identical genetic match to a chosen individual.
Christopher Shaw, Professor of Neurology and Neurogenetics at King’s College London and Director, Maurice Wohl Clinical Neuroscience Institute, said:
“This is an important advance because it is feasible – one embryonic stem cell line was generated from just two eggs. It also provides an alternative and more physiological method of reprogramming. Like many good experiments caffeine has made an invaluable contribution.”
Prof Mary Herbert, Professor of Reproductive Biology, Institute for Aging and Health at Newcastle University, said:
“This interesting report from the Oregon-based Mitalipov research group claims to have overcome a longstanding barrier to successful reprogramming of somatic cells by human oocytes. Provided that the experiments are reproducible in the hands of others, the findings offer the potential to accelerate progress towards the development of patient-specific embryonic stem cells to treat a range of degenerative diseases.”
Prof Alison Murdoch, Head of Newcastle Fertility Centre at Life, Newcastle University, said:
“The importance of the egg donor is again illustrated in this paper. Only high quality human eggs had the potential to reprogram somatic cells. It is remarkable that adding caffeine was the key that resulted in ES cell lines from all 3 donors.”
Dr Paul De Sousa, Reader, Centre for Regenerative Medicine, University of Edinburgh, and Chief Scientist, Roslin Cells Ltd., said:
“Fifteen years ago, the creation of Dolly the sheep by adult somatic cell nuclear transfer (SCNT) into an egg, and the derivation of human embryo stem cells, by the groups of Ian Wilmut and Jamie Thomson in the UK and US, respectively, inspired a vision for human “therapeutic cloning” whereby person specific embryo stem cells with a “pluri”-potency to make all of the cells of the human body could be created to model and treat human diseases. Realising this aspiration was challenged by the inefficiency of the SCNT process in animals and humans and the limited availability of human eggs for research. Almost a decade later scientific and public interest in SCNT was shifted by the ability to induce a state of pluripotency in adult cells by gene transfer, first reported by the laboratory of Shinya Yamanaka in Japan. This achievement also helped diffuse public concerns for the development of SCNT as a means for reproduction. This report from the group of Shoukhrat Mitalipov now provides a convincing demonstration that human SCNT can indeed be used efficiently to create pluripotent embryo stem cells. As such it will likely stir renewed interest in research and applications that had become discounted as impractical.
“The work builds on advances in SCNT validated by many researchers in diverse animal models since the original Dolly experiments, coupled with over a decade of committed effort of the group itself in primate and human SCNT and embryo stem cell derivation. The group’s success is attributed to optimisation of the component steps in the SCNT process beginning with egg quality and involving gentle forms of physical treatment and drugs to tightly control the onset of development during manipulation. Significantly, modest numbers of eggs recoverable from a single donor after hormonal stimulation were reproducibly sufficient to produce SCNT embryos and embryo stem cell lines with comparable efficiency and properties as can be obtained from fertilised embryos. Further research is required to assess how these cells will compare with those produced by gene transfer, although the comparative ease with which the latter are created means they are unlikely to be replaced any time soon. However, this achievement will undoubtedly renew interest not to mention concern for the use of eggs and SCNT for research and reproductive purposes. The remarkable and singular capacity of egg cells to “reprogram” developmental competence, provides valuable opportunities to identify the key factors which underpin this process. This knowledge alone would be valuable to both stem cell research and the treatment of infertility. This research can also underpin the development of new medical interventions to prevent the maternal transmission of mitochondrial diseases through embryo pronuclear transfer, which the group are also expert in. This application is distinct from the use of SCNT to produce children derived from an adult cell donor, although all such interventions demand comprehensive ethical and medical scrutiny to ensure that the risk of harm does not outweigh the intended benefit.”
Professor Robin Lovell-Badge, Head of Developmental Genetics, MRC National Institute for Medical Research, said:
“This interesting work at last brings the topic of therapeutic cloning in humans back into the realm of good science rather than controversy.
“This approach was proposed about 15 years ago as a way to derive patient-specific ES cell lines that can be used as a tool for research into the underlying causes and development of genetic diseases, and perhaps to derive treatments for these. However, although there have been proof-of-principle experiments using mice and monkeys, and several false claims and failed attempts, until now no one had been able to carry out the whole procedure with human somatic cells (such as skin cells) and human eggs to give ES cell lines (which have the ability to make any cell type in the body) that are genetically identical to the somatic cell donor.
“The scientists responsible for the latest work managed to overcome many of the technical problems experienced previously by others, making each step more efficient, and found that therapeutic cloning could work very successfully. This means that although early human embryo development is slightly different from that of other animals, including monkeys, we are not that unique.
“Patient-specific ES cell lines made in this way can now be compared with similar cells made by other more recent methods that don’t require eggs and cloning (so-called iPS, or induced pluripotent cells), but which may carry mutations and other abnormalities that could compromise their clinical application.
“With two methods available, it is exciting times for scientists trying to link underlying genetic causes to disease.”
For further information on many of the processes involved see SMC resources:
Science Media Centre guide to embryonic stem cells
Briefing notes on human stem cells
Embryonic stem cells and nuclear reprogramming
Additional details and explanations from Professor Lovell-Badge:
“It always seemed strange that SCNT and subsequent derivation of embryonic stem cell lines was relatively efficient in other mammals, but unachievable in humans. There was no reason to think that human eggs lacked the ability to reprogramme an incoming somatic cell nucleus – indeed that this was possible had been shown previously by others, although apparently not in a way that would allow ES cell derivation. So what was wrong? Was it just a problem of efficiency and therefore a problem for researchers to obtain sufficient numbers of human eggs?
“The lab of Shoukrat Mitalipov set about refining many of the technical steps used in SCNT, beginning with their experience of this with non-human primates (macaque monkeys) with which they had previously been successful. It seems that several of these steps were simply sub-optimal for human eggs.
“The most critical problem, which they had recognised in an earlier paper this year (dealing with related techniques for avoiding mitochondrial disease), was to do with “activation” of the egg. This is the process that initiates embryo development and it is normally triggered by the sperm when it fuses to the egg. (Activation involves the release of calcium ions that are stored in vesicles within the egg, which then stimulate a range of biochemical reactions.) For proper development after SCNT it is essential that this occurs just at the right time – not too early and not too late. The authors could fix the latter problem, by using an electric shock (referred to in the paper as an electroporation stimulus) alone, rather than in combination with other agents usually employed to help activation. This was used to activate the egg at an appropriate time after the somatic cell (a skin fibroblast) has been fused into the egg that has had its own nuclear DNA removed.
“However, early activation was the bigger problem. Simply removing the “spindle” (the structure within the egg containing the nuclear DNA) often led to premature activation. The authors hypothesise that this leads to loss of the (as yet unknown) reprogramming factors that are present in the egg cytoplasm. They managed to solve this by maintaining the eggs in medium with a low level of caffeine. I generally need a cup of tea or coffee to activate myself, but in this case caffeine prevents activation of the egg happening prematurely.
“Using this and other modifications to the methods, the authors were able to have very efficient development to blastocyst stages (the stage prior to implantation, where the embryo has around 100 cells of just two or three types). And critically, these were very efficient at giving rise to embryonic stem cell lines in vitro. These cell lines had identical nuclear DNA to the original somatic cells used for SCNT, but mitochondrial DNA mostly corresponding to that of the egg donor, as expected.
“SCNT would allow the derivation of patient-specific ES cell lines that can be used as a tool for research into the causes and development of genetic diseases. They could also be used for drug screening, and for other types of personalised medicine, potentially including transplants for cell-based therapy.
“However, another method of obtaining essentially the same type of cells, termed iPS cells, has now been used successfully for a number of years towards these aims. This is a relatively simple method, that does not need human eggs or complex equipment and expertise, which uses specific factors to directly reprogramme somatic cells from a patient into induced pluripotent stem cells, that are ES-like in their properties. So why is it still important to have SCNT-derived ES cells? The direct reprogramming methods (of which there are now many) are relatively inefficient, but more critically there is evidence that each iPS cell line may have accumulated a set of mutations and/or epigenetic changes that might affect their properties in vitro or after transplant back into a patient. Most regulatory authorities consider this to be a problem, especially for the latter. It will be interesting to do a direct comparison between SCNT-ES cells and iPS cells from the same individual, which the current work now makes possible.
“For those concerned about “reproductive cloning”, the efficiency to obtain blastocyst stage embryos and ES cells, does not mean that implanted embryos would develop normally. Many years of experience with reproductive cloning in animals tells us that the vast majority of cloned embryos fail at some point during gestation, most early after implantation, but all the way up to birth and beyond. It is an unsafe procedure in animals and it will similarly be an unsafe procedure in humans. For this reason alone it should not be attempted. Moreover, while there are good arguments for SCNT to obtain ES cells (“therapeutic cloning”) there are no valid reasons for attempting to carry out reproductive cloning in humans. We are not just a product of our DNA, which is the only thing that is copied in cloning. Nurture and environment are at least as important in determining who we are, therefore cloning cannot be used to bring back a loved one.
“Of more immediate importance, the same authors have published on using similar techniques as a way to avoid mitochondrial disease. This would involve transferring the spindle (with the nuclear DNA) from an egg carrying mutant mitochondria into an egg with normal mitochondria from which its own spindle has been removed (a technique termed Maternal Spindle Transfer or MST). This does not involve any form of cloning as the egg will be fertilised with sperm from the father. However, some of the methods adopted in the current paper may overcome the problems of efficiency, especially those due to abnormal egg activation.”
“The idea of using caffeine came from previous experiments they had performed with monkey eggs. Caffeine inhibits certain protein phosphatase enzymes that are involved in the degradation of “maturation promoting factor (MPF)”, a factor that is essential for controlling the cell cycle machinery in the egg.
“The authors also changed the method used to introduce the somatic cell nucleus into the egg. They used the coat (envelope) from a virus that is very efficient at promoting fusion of cell membranes to fuse the skin cell into the egg (this is making use of the mechanism the virus would normally use to get into a cell, but there is no viral genome present, so the eggs are not infected), followed by an electric shock (referred to in the paper as an electroporation stimulus) to activate the egg.
“Ionomycin is a drug that makes very small holes in cell membranes allowing Calcium ions to move through them. This is often used in activation protocols to release Calcium ions from the vesicles in which they are stored in the egg.
“The authors also made use of trichostatin A (TSA) a factor that generally releases repressed gene activity, which is thought to help reprogramming of the somatic cell – however, they found that too much of this is bad, so they carefully titrated this to the lowest levels compatible with deriving NT-ES cells.
“Ovarian stimulation to give high oocyte numbers gave eggs that were less likely to work for SCNT. The protocols for SCNT worked best when fewer eggs were produced per donor woman. Interestingly, however, some donors gave eggs that worked well, whereas those from others did not. The authors suggest a genetic influence, but this is very speculative and many other aspects of the donor’s way of life and treatment could be relevant.
“N.B. For those attempting to read all the detail in the paper, there is a problem with some of the supplementary figures, which don’t seem to match the description given in the main text.”
Additional commentary from scientists:
The paper from the Oregon group reports another milestone in the quest for patient-specific ES cells lines.
In theory, patient-specific ES cells can be generated by inducing somatic cells to revert to an embryonic state either by genetic manipulation to produce so called induced pluripotent stem cells (iPSCs), or by performing somatic cell nuclear transfer (SCNT) to harness the reprogramming power of the oocyte (the female gamete).
The few studies published on human SCNT indicate that human oocytes reprogramme somatic cells less efficiently than those of other species. A study published by the Egli group in 2011 indicated that this was due to a requirement for factors associated with, or encoded by the oocyte chromosomes, into which nuclear DNA is packaged. Having tried and failed to derive ES cells using the conventional procedure of fusing a somatic cell with an enucleated oocyte, they successfully derived ES cells by using intact instead of enucleated oocytes. While this study gave ground to a plausible framework for understanding the biological barriers to efficient reprogramming in human oocytes, the ES cell lines contained three sets of chromosomes instead of two and therefore did not provide an immediate route to SCNT-based cell therapies.
The latest addition to the field from the Oregon group indicates that efficient reprogramming can be achieved by exposing oocytes to caffeine during the SCNT procedure. Caffeine has a number of effects at the cellular level, one of which is to inhibit the calcium signals responsible for triggering the onset of embryonic development. The authors concluded that premature onset of embryonic development is the major barrier to reprogramming of somatic cells in human oocytes. By using caffeine to prevent this, they derived ES cells without the help of the oocyte nuclear genetic material.
The development of SCNT techniques to reproducibly derive ES cells is significant for a number of reasons. In the context of iPSCs, SCNT-derived ES cells are thought to provide a gold standard against which different approaches to generating iPSCS can be evaluated. In this regard, it is interesting to note that the somatic cells used by the Oregon group were obtained from open cell repositories. It should therefore be possible to use these same somatic cells to generate iPSCs for direct comparison with the Oregon SCNT-derived ES cell lines. This may advance progress by helping to inform the development of iPSC procedures that closely mimic the reprogramming activity of oocytes.
The ultimate aim of SCNT is to develop patient-specific ES cells for the treatment of degenerative diseases. Of particular interest are degenerative disease caused by mutations in mitochondrial DNA, which is present in several thousand copies per cell. These diseases are unlikely to be amenable to iPSC-based technologies due to persistence of the mitochondrial DNA mutations present in the somatic cells. SCNT, on the other hand, is likely to result in dilution of the mutation by the vast amount of mitochondrial DNA present in the oocyte.
This use of SCNT to treat mitochondrial DNA disease should not be confused with the technologies of pronuclear transfer and spindle transfer currently being developed to prevent transmission of mitochondrial DNA mutations from a mother to her child. These techniques involve transfer of the nuclear genetic material between oocytes either before or after fertilisation. These techniques do not share the biological complexities of somatic cell reprogramming required for successful SCNT. Importantly, the ultimate aim of pronuclear transfer and spindle transfer is to produce a healthy baby, free of mitochondrial DNA disease. By contrast, the ultimate aim of SCNT is to produce ES cell lines for the purposes of treating patients who already suffer from a variety of degenerative diseases, including, potentially, mitochondrial DNA disease.
‘Human embryonic stem cells derived by somatic cell nuclear transfer’ by Shoukhrat Mitalipov et al. published in Cell on Wednesday 15th May.