[caption id="attachment_23123" align="alignnone" width="800"] Nuclear extraction of a donor egg. The first image shows the egg cell (the large circular object being held in place by a pipette to the left.) Later shots show the egg nucleus being extracted from the egg cell (by a different pipette) — all in preparation for its fusion with a skin cell to kick off the cloning process. Click to enlarge. (Photos courtesy of Oregon Health & Science University)[/caption] [caption id="attachment_23112" align="alignleft" width="389"] Senior study author Shoukhrat Mitalipov, PhD, in his lab at Oregon Health & Science University[/caption] Somatic cell nuclear transfer (SCNT) is a technique in which the nucleus of a donor cell is transferred to an egg cell whose nucleus has been removed, generating embryos that are almost an identical genetic match to the donor individual. For the first time, a team of scientists has used SCNT to produce human embryonic stem cells (hESCs).
This milestone, published today by Cell Press in the journal Cell, opens up new avenues for using stem cells to understand patient-specific causes of disease and for developing personalized therapies.
“Our finding offers new ways of generating stem cells for patients with dysfunctional or damaged tissues and organs,” said senior study author Dr. Shoukhrat Mitalipov of Oregon Health & Science University. “Such stem cells can regenerate and replace those damaged cells and tissues and alleviate diseases that affect millions of people.”
Current technology is problematicAnother technique that has been used to generate patient-specific stem cells to model diseases is called induced pluripotent stem cells (iPS). It uses cells that are generated directly from the patient’s somatic cells by adding a cocktail of cellular factors to stimulate regression to a stem cell state. However, concerns that this technique may generate unexpected mutations in the stem cells means that researchers are still keen to find ways to generate hESCs by other means.
In the past, researchers have used SCNT to generate only mouse and monkey embryonic stem cells — immature cells that can develop into different types of specialized cells, from neurons to heart muscle cells. Most previous attempts failed to produce human SCNT embryos that could progress beyond the 8-cell stage, falling far short of the 150-cell blastocyst stage that could provide hESCs for clinical purposes. Until now, it was not clear which factors and protocols are important for promoting SCNT embryonic development.
How the new approach works[caption id="attachment_23078" align="alignright" width="478"] Somatic cell nuclear transfer (SCNT)[/caption] To overcome these hurdles, Dr. Mitalipov and his team started in familiar territory, refining methods for producing monkey SCNT embryos. Using these optimized protocols, they transferred nuclei from human skin cells into the cytoplasm of human egg cells, generating blastocysts that gave rise to hESC colonies. The resulting hESCs resembled those derived from fertilized embryos, had no chromosomal abnormalities, showed normal gene activity, and were capable of turning into more specialized cell types that could be used for replacing damaged tissues.
Surprisingly, the best outcomes came from donors who produced a low number of high-quality egg cells. “It was thought that, to make human SCNT work, many thousands of human eggs would be needed,” Dr. Mitalipov said. “We were able to produce one ESC line using just two human eggs, which would make this approach practical for widespread therapeutic use.” [note color="#f1f9fc" position="alignnone" width=800 margin=10]
View the articleThe article is freely available on Cell.com until June 20: “Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer” (dx.doi.org/10.1016/j.cell.2013.05.006)
Learn moreFor information on many of the processes involved, the Science Media Centre has prepared the following resources:
Dr. Shoukhrat Mitalipov talks about his work[divider]
What experts are sayingThe Science Media Centre interviewed experts in the UK who were not affiliated with this research about its implications. Here are their responses. Dr. Mary Herbert, Professor of Reproductive Biology at the Institute for Aging and Health at Newcastle University:
This interesting report ... 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.Dr. Alison Murdoch, Professor of Reproductive Medicine and Head of Newcastle Fertility Centre at Life, Newcastle University:
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, Senior Research Fellow and Project Manager at the Centre for Regenerative Medicine, University of Edinburgh, and Chief Scientist, Roslin Cells:
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.Dr. Robin Lovell-Badge, Head of Developmental Genetics, MRC National Institute for Medical Research:
“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.”[divider] [caption id="attachment_18867" align="alignleft" width="150"] Mary Beth O'Leary[/caption]