Mitochondrial Donation: How Many Women Could Benefit?

Two and a Half Thousand Women Could Benefit from Mitochondrial Donation in the UK

Thursday, 29 January 2015

Almost 2,500 women of child-bearing age in the UK are at risk of transmitting mitochondrial disease to their children, according to the most recent estimates published today in the New England Journal of Medicine.

The research offers the most recent evidence yet of how many families could potentially be helped by new IVF techniques to prevent mitochondrial disease, which would be permitted by new regulations on which a vote in parliament is imminent.

Mitochondrial diseases are caused by inherited mutations in the DNA contained in mitochondria - tiny structures present in every cell that generate energy. Mitochondrial diseases can be devastating and particularly affect tissues that have high energy demands - brain, muscle (including heart), liver and kidney.

New IVF-based techniques have been developed which have the potential to prevent the transmission of serious mitochondrial disease. Known as 'mitochondrial donation' the techniques involve removing faulty mitochondria inherited from the mother and replacing them with the healthy mitochondria of another woman. The nuclear DNA, containing 99.9% of genetic material from the mother and father, remains unchanged.

Researchers at the Wellcome Trust Centre for Mitochondrial Research at Newcastle University, which will be the first to offer mitochondrial donation if parliament agrees to new regulations of the Human Fertilisation and Embryology Act (1990), have now calculated how many women have disease-causing mutations in their mitochondrial DNA in order to estimate how many could potentially benefit. The new regulations only allow for mitochondrial donation to prevent mitochondrial disease and set no precedent for genetic manipulation of nuclear DNA.

They calculate that 2,473 women in the UK, and 12,423 women in the US, aged between 15 and 44 years, are at risk of passing on potentially lethal mitochondrial DNA disease to their children. This equates to an average of 152 births per year in the UK, and 778 births per year in the US.

The estimates were made by identifying the number of women in North East England who are at risk of passing on mitochondrial disease to their children and extrapolating the figure to the rest of the UK, based on the relative number of women of child-bearing age in the North East compared to the UK as a whole. A similar method was used for the US figures. The study did not account for variance due to ethnicity or potentially different fertility rates in different parts of the UK.

Researchers also assessed the fertility of women with mitochondrial DNA mutations. To do this they compared fertility data from their patients' to data about the general population, obtained from the UK Office for National Statistics. They found that mitochondrial mutation has no statistically significant effect on fertility rate.

Dr Gráinne Gorman from the Wellcome Trust Centre for Mitochondrial Research at Newcastle University, and joint first author of the paper, said:

"Our estimate of how many women could benefit from mitochondrial donation is based on our data from North East England, where we have very detailed insight into how many women are affected. We are confident that there are a similar number of women across the UK at risk of passing on mitochondrial disease to their children."

Professor Doug Turnbull, Director of the Wellcome Trust Centre for Mitochondrial Research at Newcastle University, and an author of the paper, said:

"Our findings have considerable implications for all countries that are considering allowing mitochondrial donation techniques. In the UK we are waiting for parliament to decide whether to support these regulations. This would allow women who carry these mutations greater reproductive choice. "

Source: Wellcome Trust 

Contact: Clare Ryan 

Reference:

Mitochondrial Donation: How many women could benefit? 

Gráinne S. Gorman, John P. Grady, Yi Ng, Andrew M. Schaefer, Richard J. McNally, Patrick F. Chinnery, Patrick Yu Wai Man, Mary Herbert, Robert W. Taylor, Robert McFarland, and Doug M. Turnbull

New England Journal of Medicine, January 28, 2015 DOI: 10.1056/NEJMc1500960

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Scientists find that SCNT Derived Cells and iPS Cells are Similar

Scientists find that SCNT Derived Cells and iPS Cells are Similar

Thursday, 06 November 2014

A team led by New York Stem Cell Foundation (NYSCF) Research Institute scientists conducted a study comparing induced pluripotent stem (iPS) cells and embryonic stem cells created using somatic cell nuclear transfer (SCNT). The scientists found that the cells derived from these two methods resulted in cells with highly similar gene expression and DNA methylation patterns. Both methods also resulted in stem cells with similar amounts of DNA mutations, showing that the process of turning an adult cell into a stem cell introduces mutations independent of the specific method used. This suggests that both methods of producing stem cells need to be further investigated before determining their suitability for the development of new therapies for chronic diseases.

The NYSCF Research Institute is one of the only laboratories in the world that currently pursues all forms of stem cell research including SCNT and iPS cell techniques for creating stem cells. The lack of laboratories attempting SCNT research was one of the reasons that the NYSCF Research Institute was established in 2006.

"We do not yet know which technique will allow scientists to create the best cells for new cellular therapies," said Susan L. Solomon, NYSCF CEO and co-founder.

"It is critical to pursue both SCNT and iPS cell techniques in order to accelerate research and bring new treatments to patients."

While both techniques result in pluripotent stem cells, or cells that can become any type of cell in the body, the two processes are different. SCNT consists of replacing the nucleus of a human egg cell or oocyte with the nucleus of an adult cell, resulting in human embryonic stem cells with the genetic material of the adult cell. In contrast, scientists create iPS cells by expressing a few key genes in adult cells, like a skin or blood cell, causing the cells to revert to an embryonic-like state. These differences in methods could, in principle, result in cells with different properties. Advances made earlier this year by NYSCF Research Institute scientists that showed that human embryonic stem cells could be derived using SCNT revived that debate.

"Our work shows that we now have two methods for the generation of a patient's personal stem cells, both with great potential for the development of treatments of chronic diseases. Our work will also be welcome news for the many scientists performing basic research on iPS cells. It shows that they are likely working with cells that are very similar to human embryonic stem cells, at least with regard to gene expression and DNA methylation. How the finding of mutations might affect clinical use of stem cells generated from adult cells is the subject of an ongoing debate," said Dr. Dieter Egli, NYSCF Senior Research Fellow, NYSCF - Robertson Investigator, Assistant Professor in Pediatrics & Molecular Genetics at Columbia University, and senior author on the paper.

The study, published today in Cell Stem Cell, compared cell lines derived from the same sources using the two differing techniques, specifically contrasting the frequency of genetic coding mutations seen and measuring how closely the stem cells matched the embryonic state through the analysis of DNA methylation and of gene expression patterns. The scientists showed that both methods resulted in cell types that were similar with regard to gene expression and DNA methylation patterns. This suggested that both methods were effective in turning a differentiated cell into a stem cell.

The scientists also showed that cells derived using both SCNT and iPS techniques showed similar numbers of genetic coding mutations, implying that neither technique is superior in that regard. A similar number of changes in DNA methylation at imprinted genes (genes that are methylated differentially at the maternal versus the paternal allele) were also found. It is important to note that both types of techniques led to cells that had more of these aberrations than embryonic stem cells derived from an unfertilized human oocyte, or than embryonic stem cells derived from leftover IVF embryos. These findings suggest that a small number of defects are inherent to the generation of stem cells from adult differentiated cells and occur regardless of the method used.

Source: New York Stem Cell Foundation

Contact: David McKeon

Reference:

Comparable Frequencies of Coding Mutations and Loss of Imprinting in Human Pluripotent Cells Derived by Nuclear Transfer and Defined Factors

Bjarki Johannesson, Ido Sagi, Athurva Gore, Daniel Paull, Mitsutoshi Yamada, Tamar Golan-Lev, Zhe Li, Charles LeDuc, Yufeng Shen, Samantha Stern, Nanfang Xu, Hong Ma, Eunju Kang, Shoukhrat Mitalipov, Mark V. Sauer, Kun Zhang, Nissim Benvenisty, Dieter Egli

Cell Stem Cell, Volume 15, Issue 5, p634–642, 6 November 2014, DOI: http://dx.doi.org/10.1016/j.stem.2014.10.002

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Some Stem Cell Methods Closer to "Gold Standard" than Others

Nuclear transfer appears superior for creating embryonic stem cells

Thursday, 03 July 2014

Researchers around the world have turned to stem cells, which have the potential to develop into any cell type in the body, for potential regenerative and disease therapeutics.

Now, for the first time, researchers at the Salk Institute, with collaborators from Oregon Health & Science University and the University of California, San Diego, have shown that stem cells created using two different methods are far from identical. The finding could lead to improved avenues for developing stem cell therapies as well as a better understanding of the basic biology of stem cells.

The researchers discovered that stem cells created by moving genetic material from a skin cell into an empty egg cell — rather than coaxing adult cells back to their embryonic state by artificially turning on a small number of genes — more closely resemble human embryonic stem cells, which are considered the gold standard in the field.

Joseph R. Ecker, Professor, Genomic Analysis

Laboratory. Credit: Courtesy of the Salk

Institute for Biological Studies. 

"These cells created using eggs' cytoplasm have fewer reprogramming issues, fewer alterations in gene expression levels and are closer to real embryonic stem cells," says co-senior author Joseph R. Ecker, professor and director of Salk's Genomic Analysis Laboratory and co-director of the Center of Excellence for Stem Cell Genomics. The results of the study were published today in Nature.

Human embryonic stem cells (hESCs) are directly pulled from unused embryos discarded from in-vitro fertilization, but ethical and logistical quandaries have restricted their access. In the United States, federal funds have limited the use of hESCs so researchers have turned to other methods to create stem cells. Most commonly, scientists create induced pluripotent stem (iPS) cells by starting with adult cells (often from the skin) and adding a mixture of genes that, when expressed, regress the cells to a pluripotent stem-cell state. Researchers can then coax the new stem cells to develop into cells that resemble those in the brain or in the heart, giving scientists a valuable model for studying human disease in the lab.

Over the past year, a team at OHSU built upon a technique called somatic cell nuclear transfer (the same that is used for cloning an organism, such as Dolly the sheep) to transplant the DNA-containing nucleus of a skin cell into an empty human egg, which then naturally matures into a group of stem cells.

Shoukhrat Mitalipov, Ph.D., Oregon Health &

Science University, led a team that found that a

process called "somatic cell nuclear transfer" is

much better and more accurate at

reprogramming human skin cells to become

embryonic stem cells. Credit: Oregon Health &

Science University.

Ecker, holder of the Salk International Council Chair in Genetics, teamed up with Shoukhrat Mitalipov, developer of the new technique and director of the Center for Embryonic Cell and Gene Therapy at OHSU, and UCSD assistant professor Louise Laurent to carry out the first direct comparison of the two approaches. The scientists created four lines of nuclear transfer stem cells all using eggs from a single donor, along with seven lines of iPS cells and two lines of the gold standard hESCs. All cell lines were shown to be able to develop into multiple cell types and had nearly identical DNA content contained within them.

But when they looked closer at the cells, the researchers spotted some differences: the patterns of methylation — chemical flags that are added to genes to control their expression — varied between the cell lines. This indicates a difference in how and when genes, despite having identical sequences, might be expressed. The methylation of nuclear transfer cells more closely resembled hESCs than the iPS cells did. And when the investigators looked at patterns of actual gene expression — by measuring the levels of particular RNA strands produced by each cell — the differences continued. Once again, nuclear transfer cells had RNA levels closer to embryonic cells, making them more accurate for basic research and therapeutic studies.

"Both the DNA methylation and gene expression data show that nuclear transfer does a better job at erasing the signature of the original skin cell," says Laurent, who is a co-senior author of the paper.

"If you believe that gene expression is important, which we do, then the closer you get to the gene expression patterns of embryonic stem cells, the better," Ecker says.

"Right now, nuclear transfer cells look closer to the embryonic stem cells than do the iPS cells."

Ecker doesn't expect labs to race to make the switch to nuclear transfer protocols — after all, the method falls within those restricted for federal funding. But he thinks the new observation likely holds lessons that could help improve the protocols for making iPS cells.

"What this is telling us is that you can use the standard mix of genes and they do a pretty good job of creating iPS cells," Ecker says.

"But they're not perfect. The material in an egg does a better job than just those four genes alone."

If researchers can pin down what it is within an egg that drives the production of pluripotent stem cells, they may be able to integrate that knowledge into iPS methods to improve stem cell therapy for disease.

"At this point, nuclear transfer stem cells combine the key advantages of both hESCs and iPS cells and, as such, are ideal for clinical applications in regenerative therapy," adds Mitalipov.

Other researchers on the study were Ryan C. O'Neil, Yupeng He, Matthew D. Schultz, Manoj Heriharan, Joseph R. Nery, and Rosa Castanon of the Salk Institute for Biological Studies; Hong Ma, Brittany Daughtry, Masahito Tachibana, Eunju Kang, Rebecca Tippner-Hedges, Riffat Ahmed, Nuria Marti Gutierrez, Crystal Van Dyken, Alimujiang Fulati, Atsushi Sugawara, Michelle Sparman, Paula Amato and Don P. Wolf of Oregon Health & Science University; Robert Morey, Karen Sabatini and Rathi D. Thiagarajan of the University of California, San Diego; and Sumita Gokhale of the Boston University School of Medicine.

Source: Salk Institute for Biological Studies

Contact: Kristina Grifantini

Reference:

Abnormalities in human pluripotent cells due to reprogramming mechanisms

Hong Ma, Robert Morey, Ryan C. O'Neil, Yupeng He, Brittany Daughtry, Matthew D. Schultz, Manoj Hariharan, Joseph R. Nery, Rosa Castanon, Karen Sabatini, Rathi D. Thiagarajan, Masahito Tachibana, Eunju Kang, Rebecca Tippner-Hedges, Riffat Ahmed, Nuria Marti Gutierrez, Crystal Van Dyken, Alim Polat, Atsushi Sugawara, Michelle Sparman, Sumita Gokhale, Paula Amato, Don P.Wolf, Joseph R. Ecker, Louise C. Laurent & Shoukhrat Mitalipov

Nature (2014), doi:10.1038/nature13551

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Three Parents and a Baby

Scientists advise caution with regard to artificial insemination method

Wednesday, 18 June 2014

Already a few dysfunctional mitochondria (in

yellow on top of the picture) could cause a

disease by overgrowing functional ones (in blue).

Credit: Illustration: Iain Johnston. 

Mitochondria are cell organelles located within animal and human cells. They produce energy for the organism, possess their own genetic material - mitochondrial DNA (mtDNA) - and are transmitted exclusively by the mother. Depending on their activity and tasks, different numbers of mitochondria are present in a cell - usually a few hundred to a thousand per body cell.

Inherited mitochondrial disorders or so-called mitochondropathies occur in about one of 10,000 humans throughout the world. Diseases such as diabetes, stroke, cardiac defects, epilepsy, or muscle weakness may originate from mitochondrial defects. Inherited mitochondrial disorders have been incurable so far. Therefore, efforts are now being made to enable women with this disease to bear healthy children by means of nuclear transfer.

Mitochondria multiply at different rates

Jörg Burgstaller, a scientist and member of Gottfried Brem's research group at the Vetmeduni Vienna, has been working for several years on the genetics of mitochondria. It was known before that different types of mitochondria within a cell can proliferate at different rates. However, it was not known whether this is a singular phenomenon or if these cases occur more frequently.

Burgstaller investigated this in four newly bred mouse models which carried different mixtures of mitochondria whose DNA were related to each other to a differing extent.

This meant no health problem for the mice since all mtDNAs are were fully functional.

The outcome was: the more distantly two types of mitochondria within an egg cell were related, the more frequently a growth advantage was noted in favour of one of the two types of mitochondria. When two different mtDNAs were equally common in cells of an organ at the time of birth, one type was completely lost after a while. One mitochondria variant had thus achieved a growth advantage compared to the other variant and superseded the latter. This effect was almost non-existent in genetically very similar mitochondria within the cells; the ratio between the two types of mitochondria was not altered in that case.

The effect is of significance in reproduction medicine

Burgstaller's results may have effects on the planned introduction of the so called "Three-Parent Baby" in Great Britain. Experts take the cell nucleus of one human egg cell whose mitochondria have a defect and place it in an egg cell with "healthy" mitochondria. The baby resulting from this procedure has three parents, namely the mother whose cell nucleus is used, the mother whose mitochondria are involved, and the father whose sperm inseminated the egg cell.

However, this method raises the following problem: in every nuclear transfer, a small number of defective mitochondria are transferred into the healthy egg cell.

"So far it was believed that this minimal 'contamination' is of no consequence for the baby. However, our data show that the effect may have dramatic consequences on the health of the offspring. If the mitochondria of both mothers are genetically very different, it may have the same effects seen in the mouse model," says Burgstaller who developed the theory together with co-author Joanna Poulton, Professor of Mitochondrial Genetics at the John Radcliffe Hospital in Oxford.

"One mitochondrial type may be able to assert itself against the other. If the assertive one happens to carry the defective mtDNA, the benefit of the therapy would be jeopardized."

The solution to the "Three-Parent Baby"-problem

Burgstaller and his colleagues suggest the following solution to the problem: the mtDNA of both mothers, i.e. the donor of the nucleus and the donor of the mitochondria, should be analysed in advance and aligned to each other. So called "matching haplotypes" could prevent the dangerous effect. In the future the effect may even be utilized in a targeted manner to suppress defective mtDNA.

Source: University of Veterinary Medicine – Vienna 

Contact: Joerg Burgstaller

Reference:

mtDNA Segregation in Heteroplasmic Tissues Is Common In Vivo and Modulated by Haplotype Differences and Developmental Stage

Joerg Patrick Burgstaller, Iain G. Johnston, Nick S. Jones, Jana Albrechtová, Thomas Kolbe, Claus Vogl, Andreas Futschik, Corina Mayrhofer, Dieter Klein, Sonja Sabitzer, Mirjam Blattner, Christian Gülly, Joanna Poulton, Thomas Rülicke, Jaroslav Piálek, Ralf Steinborn and Gottfried Brem

Cell Reports. DOI:10.1016/j.celrep.2014.05.020 

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First Disease-specific Human Embryonic Stem Cell Line by Nuclear Transfer

Major step toward cell-based therapies for life-threatening diseases

Tuesday, 29 April 2014

Using somatic cell nuclear transfer, a team of scientists led by Dr. Dieter Egli at the New York Stem Cell Foundation (NYSCF) Research Institute and Dr. Mark Sauer at Columbia University Medical Center has created the first disease-specific embryonic stem cell line with two sets of chromosomes.

Embryonic stem cells from an adult with type 1

diabetes were created by transferring the nucleus

from a skin cell of the patient into a donor oocyte.

Credit: Bjarki Johannesson, NYSCF. 

As reported today in Nature, the scientists derived embryonic stem cells by adding the nuclei of adult skin cells to unfertilized donor oocytes using a process called somatic cell nuclear transfer (SCNT). Embryonic stem cells were created from one adult donor with type 1 diabetes and a healthy control. In 2011, the team reported creating the first embryonic cell line from human skin using nuclear transfer when they made stem cells and insulin-producing beta cells from patients with type 1 diabetes. However, those stem cells were triploid, meaning they had three sets of chromosomes, and therefore could not be used for new therapies.

The investigators overcame the final hurdle in making personalized stem cells that can be used to develop personalized cell therapies. They demonstrated the ability to make a patient-specific embryonic stem cell line that has two sets of chromosomes (a diploid state), the normal number in human cells. Reports from 2013 showed the ability to reprogram foetal fibroblasts using SCNT; however, this latest work demonstrates the first successful derivation by SCNT of diploid pluripotent stem cells from adult and neonatal somatic cells.

"From the start, the goal of this work has been to make patient-specific stem cells from an adult human subject with type 1 diabetes that can give rise to the cells lost in the disease," said Dr. Egli, the NYSCF scientist who led the research and conducted many of the experiments.

"By reprograming cells to a pluripotent state and making beta cells, we are now one step closer to being able to treat diabetic patients with their own insulin-producing cells."

"I am thrilled to say we have accomplished our goal of creating patient-specific stem cells from diabetic patients using somatic cell nuclear transfer," said Susan L. Solomon, CEO and co-founder of NYSCF.

"I became involved with medical research when my son was diagnosed with type 1 diabetes, and seeing today's results give me hope that we will one day have a cure for this debilitating disease. The NYSCF laboratory is one of the few places in the world that pursues all types of stem cell research. Even though many people questioned the necessity of continuing our SCNT work, we felt it was critical to advance all types of stem-cell research in pursuit of cures. We don't have a favourite cell type, and we don't yet know what kind of cell is going to be best for putting back into patients to treat their disease."

The research is the culmination of an effort begun in 2006 to make patient-specific embryonic stem cell lines from patients with type 1 diabetes. Ms. Solomon opened NYSCF's privately funded laboratory on March 1, 2006, to facilitate the creation of type 1 diabetes patient-specific embryonic stem cells using SCNT. Initially, the stem cell experiments were done at Harvard and the skin biopsies from type 1 diabetic patients at Columbia; however, isolation of the cell nuclei from these skin biopsies could not be conducted in the federally funded laboratories at Columbia, necessitating a safe-haven laboratory to complete the research. NYSCF initially established its lab, now the largest independent stem cell laboratory in the nation, to serve as the site for this research.

In 2008, all of the research was moved to the NYSCF laboratory when the Harvard scientists determined they could no longer move forward, as restrictions in Massachusetts prevented their obtaining oocytes. Dr. Egli left Harvard University and joined NYSCF; at the same time, NYSCF forged a collaboration with Dr. Sauer who designed a unique egg-donor program that allowed the scientists to obtain oocytes for the research.

"This project is a great example of how enormous strides can be achieved when investigators in basic science and clinical medicine collaborate. I feel fortunate to have been able to participate in this important project," said Dr. Sauer. Dr. Sauer is vice chair of the Department of Obstetrics and Gynecology, professor of obstetrics and gynaecology, and chief of reproductive endocrinology at Columbia University Medical Center and program director of assisted reproduction at the Center for Women's Reproductive Care.

Patients with type 1 diabetes lack insulin-producing beta cells, resulting in insulin deficiency and high blood-sugar levels. Therefore, producing beta cells from stem cells for transplantation holds promise as a treatment and potential cure for type 1 diabetes. Because the stem cells are made using a patient's own skin cells, the beta cells for replacement therapy would be autologous, or from the patient, matching the patient's DNA.

Generating autologous beta cells using SCNT is only the first step in developing a complete cell replacement therapy for type 1 diabetes. In type 1 diabetes, the body's immune system attacks its own beta cells; therefore, further work is underway at NYSCF, Columbia, and other institutions to develop strategies to protect existing and therapeutic beta cells from attack by the immune system, as well as to prevent such attack.

The technique described in the report published today can also be translated for use in the development of personalized autologous cell therapies for many other diseases and conditions including Parkinson's disease, macular degeneration, multiple sclerosis, and liver diseases and for replacing or repairing damaged bones.

As part of the work, the scientists systematically analysed the factors that affect stem-cell derivation after SCNT. The reprogramming of skin cells from a type 1 diabetes patient by SCNT has long been sought, but has been challenging to achieve because of logistical difficulties in obtaining human oocytes for research, as well as an incomplete understanding of the biology of human oocytes.

The scientists found that the addition of specific chemicals, called histone deacetylase inhibitors, and an efficient protocol for human oocyte activation were critical to achieving development to the stage at which embryonic stem cells are derived. These findings are consistent with the 2013 report by Tachibana and colleagues that used foetal cells. Though the authors of the 2013 paper also performed studies with cells of an infant with Leigh syndrome, they did not demonstrate that diploid pluripotent stem cells could be derived from these cells. Because foetal cells are less mature than the cells after birth, it was critical to determine if diploid pluripotent stem cells could be derived from the cells of both infants and adults.

As an additional optimization of the SCNT protocol, the scientists found that it was important to maintain the integrity of the plasma membrane during manipulation, and that to do so, the agent used in the manipulations had to be at a low dose. The scientists applied this optimized protocol to skin cells of a male new-born and the cells of the adult patient with type 1 diabetes. From these two cell lines, the scientists produced a total of four SCNT-derived embryonic stem cell lines. All cell lines were diploid and could give rise to neurons, pancreatic cells, and cartilage, as well as various other cell types, demonstrating their pluripotency. Importantly, the cells of the type 1 diabetes patient also gave rise to insulin-producing beta cells.

Therefore, this is the first report of the derivation of diploid pluripotent stem cells from a patient. And together with a paper published this month in Cell Stem Cell by Chung et al., it is also the first report of diploid embryonic stem cell lines derived from a human after birth.

Dr. Nissim Benvenisty and his laboratory at Hebrew University of Jerusalem collaborated on this report by demonstrating that the cells produced were, in fact, embryonic stem cells by using microarrays to perform gene expression analysis of the cells.

Dr. Rudolph Leibel, a co-author and co-director with Dr. Robin Goland of the Naomi Berrie Diabetes Center, where aspects of these studies were conducted, said:

"This accomplishment is the product of an ongoing inter-institutional collaboration across scientific and clinical disciplines, supported by thoughtful philanthropy. The resulting technical and scientific insights bring closer the promise of cell replacement for a wide range of human disease."

NYSCF continues pursuing SCNT research despite many scientific obstacles and in light of the advent of induced pluripotent stem (iPS) cells, as it is not yet clear which type of stem cells will prove best for personalized treatments. Many thought that iPS cells, first created from human cells in 2007, would replace the need for patient-specific embryonic stem cells because they allow patient- and disease-specific stem cell lines to be generated by genetically reprogramming adult cells into becoming pluripotent cells. However, it is not clear how similar iPS cells are to naturally occurring embryonic stem cells, which remain the gold standard, and what will be the preferred cell type for therapies.

Though it is now possible to derive stem cell lines with a patient's genotype using iPS technology, the generation of stem cells using oocytes may have an advantage for use in cell replacement for diseases such as type 1 diabetes. The generation of pluripotent stem cell lines by SCNT uses human oocytes, while iPS cells use recombinant DNA, RNA, or chemicals, each of which requires its own safety testing and approval for clinical use. Human oocytes are already used routinely around the world to generate clinically relevant cells. The generation of pluripotent stem cell lines using human oocytes may therefore be particularly suitable for the development of cell-replacement therapies. Therefore, this work brings the scientists a significant step closer to this goal.

Drs. Mitsutoshi Yamada and Bjarki Johannesson, postdoctoral fellows at the NYSCF Research Institute, were the co-first authors of the paper.

Source: New York Stem Cell Foundation

Contact: David McKeon

Reference:

Human oocytes reprogram adult somatic nuclei of a type 1 diabetic to diploid pluripotent stem cells

Mitsutoshi Yamada, Bjarki Johannesson, Ido Sagi, Lisa Cole Burnett, Daniel H. Kort, Robert W. Prosser, Daniel Paull, Michael W. Nestor, Matthew Freeby, Ellen Greenberg, Robin S. Goland, Rudolph L. Leibel, Susan L. Solomon, Nissim Benvenisty, Mark V. Sauer& Dieter Egli

Nature 28 April 2014, doi:10.1038/nature13287

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Therapeutic Cloning Gets a Boost with New Research Findings

San Antonio and Honolulu researchers make important discoveries about point mutation rates in cloned mouse foetuses Thursday, 26 March 2009 Germ cells, the cells which give rise to a mammal's sperm or eggs, exhibit a five to ten-fold lower rate of spontaneous point mutations than adult somatic cells, which give rise to the body's remaining cell types, tissues and organs. Despite their comparatively higher mutation rates, however, adult somatic cells are used as the donor cells in a cloning process called somatic cell nuclear transfer (SCNT). This made researchers wonder if cloning by SCNT leads to progeny with more mutations than their naturally conceived counterparts. Also, would cloned foetuses receive DNA programming predisposing them to develop mutations faster than natural foetuses of the same age? Those scenarios are simply not likely, say researchers at the University of Texas at San Antonio, The University of Texas Health Science Center at San Antonio and The University of Hawaii at Honolulu's John A. Burns School of Medicine. The team, which spent more than five years analyzing mutation rates and types in cloned Big Blue® mouse foetuses recently published its findings in the online Early Edition of the Proceedings of the National Academy of Sciences in a paper titled "Epigenetic regulation of genetic integrity is reprogrammed during cloning." The paper offers the first direct demonstration that cloning does not lead to an increase in the frequency of point mutations. John McCarrey, professor of cellular and molecular biology at UTSA and the study's principal investigator, suggests a "bottleneck effect" is partially responsible for the observations his team recorded. "To create a cloned foetus by somatic cell nuclear transfer, only one adult somatic cell – one donor cell – is needed," he explains. "Because a random cell population exhibits a low mutation rate overall and only one cell from that population is used for cloning, the likelihood is remote that the cell chosen to be cloned will transfer a genetic mutation to its cloned offspring. Therefore, the bottleneck effect limits the transfer of mutations from donor cells to cloned offspring." Not only did the researchers find that SCNT does not lead to an increase in the frequency of point mutations in cloned mice, the team also found that naturally conceived foetuses and cloned foetuses that are the same age have similar rates of spontaneous mutation development. They attribute this finding to epigenetic reprogramming. It is known in the scientific community that germ cells contain an epigenome, a programmed state of the genome, which keeps mutation rates low. They suggest this type of epigenome is found in germ cells because those cells are responsible for contributing genetic information to subsequent generations. Adult somatic cells (the donor cells in SCNT) have higher mutation rates and less stringent epigenetic programming to avoid mutations than germ cells. Offspring produced from somatic cells by cloning have mutation rates similar to those in offspring produced by natural reproduction, suggesting that the epigenome of an adult somatic cell is reprogrammed during cloning to maintain the genetic integrity of that cell's progeny. Reference: Epigenetic regulation of genetic integrity is reprogrammed during cloning Patricia Murphey, Yukiko Yamazaki, C. Alex McMahan, Christi A. Walter, Ryuzo Yanagimachi, and John R. McCarrey PNAS, March 2, 2009, doi: 10.1073/pnas.0900687106 ......... ZenMaster

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Chinese Researchers Make Cloned Human Blastocysts

SCNT Using an Alternative Enucleation Method for Patient-specific Embryonic Stem Cells (ESCs) Tuesday, 03 February 2009 China Daily report that a research team at the Shandong Stem Cell Engineering Research Center has successfully cloned five human blastulas from 135 eggs on experiment, according to a press conference jointly held by the research centre and Yantai Procreation Medicine Center on Monday. The Yantai Region is located north-central on the Shandong Peninsula, south of the Bohai Sea. Of the five cloned human blastulas, four were from skin fibroblasts of healthy donors while the other one was from lymphocytes of patients with Parkinson disease. At the press release, leader of the research team, Li Jian-yuan explained the newly invented cloning technology is expected to facilitate medical treatment for patients like the sufferers of Parkinson disease. Somatic cell nuclear transfer (SCNT) was used to generate patient-specific embryonic stem cells (ESCs) from blastocysts cloned by nuclear transfer (ntESCs). In this study, a total of 135 oocytes were obtained from 12 healthy donors (30–35 years). Human oocytes, obtained within 2 h following aspiration, were enucleated and human fibroblasts or lymphocytes were used to construct the SCNT embryos. The web edition of the science journal "Cloning and Stem Cells" reported the Chinese scientific achievement on January 27, 2009. Reference: Human Embryos Derived by Somatic Cell Nuclear Transfer Using an Alternative Enucleation Approach Jianyuan Li, Xuexia Liu, Haiyan Wang, Shouxin Zhang, Fujun Liu, Xuebo Wang, Yanwei Wang. Cloning and Stem Cells. ahead of print. doi:10.1089/clo.2008.0041 ......... ZenMaster

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Animal Eggs Not Suitable Substitutes to Produce Stem Cells

Eggs of another species turns off the genes needed to make an embryo instead of turning them on Monday, 02 February 2009 Since the cloning of Dolly the Sheep over a decade ago, somatic cell nuclear transfer (SCNT) has been considered a promising way to generate human, patient-specific stem cells for therapeutic applications. The shortage of human donor eggs has led to efforts to substitute animal oocytes. However, a new study published online ahead of print in the Volume 11, Number 2, 2009 issue of Cloning and Stem Cells, demonstrates that animal oocytes lack the capacity to fully reprogram adult human cells. Robert Lanza, M.D. from Advanced Cell Technology (Worcester, MA), and colleagues compared the reprogramming of human cells using oocytes obtained from cows, rabbits, and humans. They report their findings in a paper entitled, "Reprogramming of Human Somatic Cells Using Human and Animal Oocytes." “Mixing human and animal cells does not appear to program the egg properly,” said Dr. Robert Lanza in an interview with Reuters. "For the last decade, we've carried out literally hundreds of experiments trying to create patient-specific stem cells using animal eggs," Lanza said. "We got beautiful little hybrid embryos, but it didn't work no matter how hard we tried." The ability to reprogram human cells using oocytes would enable the production of patient-specific stem cells that could then be differentiated to become any type of somatic cell and used for cell or tissue repair or placement therapy. This extensive reprogramming requires that the oocyte turn on, or up-regulate a large number of genes in the donor nucleus. Although previous reports have documented the formation of cloned embryos using both human and animal eggs, to date, there have been no data indicating to what extent the donor human DNA was reprogrammed. Lanza et al. show for the first time that human oocytes have the capacity to change these patterns of gene expression, and that interspecies (human-to-animal) cloning does not produce the same results. Although the human-bovine and human-rabbit clones looked similar to the human-human embryos, the human-animal hybrids did not exhibit the changes in gene expression seen in the human-human clones and normal embryos. A mouse-human hybrid petered out after just one division. The cow and rabbit human hybrids went further, but stopped at the point when maternal DNA is supposed to kick in and turn the ball of cells into a proper embryo, Lanza said. Lanza's team used a new method called global gene expression analysis to see which genes were turned on and off as the eggs grew. "We never had the tools before to actually look inside the cell and see what's going on," Lanza said. It appears that using the egg of another species turns off the genes needed to make an embryo instead of turning them on, he said. But the human-human clone did turn on the right genes, although it, too stopped dividing before it could produce stem cells, Lanza said. "We see exactly the same genes turned on in a normal embryo are actually turned on in a human clone," he said. “We examined the factors recently used to reprogram skin cells (to induce pluripotent stem cells),” said Robert Lanza. “At the center of cellular reprogramming lies the activation of the transcription factors Oct4, Sox2, and Nanog. These core factors were activated in both the normal and cloned human embryos. In striking contrast, the human-animal hybrids showed no difference or a down-regulation of these critical pluripotency genes −effectively silencing them — thus making the generation of stem cells impossible. Without appropriate reprogramming, these data call into question the potential use of animal egg sources to generate patient-specific stem cells. It also renders the moral controversy surrounding the use of human-animal hybrids mute.” Specifically, they did not achieve up-regulation of these critical pluripotency-associated genes needed for stem cell production. For example, human oocytes significantly up-regulated Oct-4, Sox-2, and Nanog (22-fold, 6-fold, and 12-fold, respectively), whereas the bovine and rabbit oocytes either showed no difference or a down-regulation of these critical pluripotency-associated genes, effectively silencing them. "This very important paper suggests that livestock oocytes are extremely unlikely to be suitable as recipients for use in human nuclear transfer. This is very disappointing because it would mean that production of patient-specific stem cells by this means would be impracticable," says Ian Wilmut, Ph.D., Editor-in-Chief of Cloning and Stem Cells and director of the Centre for Regenerative Medicine, in Edinburgh. Reference: Reprogramming of Human Somatic Cells Using Human and Animal Oocytes Young Chung, Colin E. Bishop, Nathan R. Treff, Stephen J. Walker, Vladislav M. Sandler, Sandy Becker, Irina Klimanskaya, Wan-Song Wun, Randall Dunn, Rebecca M. Hall, Jing Su, Shi-Jiang Lu, Marc Maserati, Young-Ho Choi, Richard Scott, Anthony Atala, Ralph Dittman, Robert Lanza Cloning and Stem Cells. ahead of print, doi:10.1089/clo.2009.0004 ......... ZenMaster

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UN-GA Ban on All Human Cloning to be Reconsidered

UN International Bioethics Committee to debate the issue once more Monday, 13 October 2008 The permissibility of therapeutic cloning will be the focus of a United Nations ethics panel later this month when it considers whether a non-binding General Assembly declaration calling on Member States to ban all forms of human cloning should be reassessed in light of scientific, ethical, social, political and legal advances. In 2005 a minority of the General Assembly declared all human cloning incompatible with human dignity and protection of life, voting 84 in favour, 34 against, 37 abstaining and 36 absent, after a decade of work on reproductive cloning by the International Bioethics Committee (IBC) of the UN Educational, Scientific and Cultural Organization (UNESCO). Now the IBC will debate the issue once more at a two-day meeting at UNESCO headquarters in Paris beginning 28 October, noting that some people, mainly scientists, are urging a different approach to therapeutic cloning. “Recent technological developments and new prospects for the use of stem cells in the therapy of human diseases have once again raised the issue of adequacy of international regulations governing this research,” an IBC working group set up at the request of UNESCO Director-General Koïchiro Matsuura said in a report in September. The report noted that the main point of controversy in the 2005 Declaration was the question of linking the issues of reproductive and non-reproductive cloning, which was not agreeable to many States who abstained or voted against. The Group calls for human reproductive cloning to be banned at the international level by a legally binding convention, while guidelines for regulating human embryo and stem cell research in countries where it is legal should be developed at the international level. An Observatory Group could be established to new ethical, legal, social, political and scientific developments, and UNESCO should develop specific strategies and materials to promote international dialogue on this topic. The other focus of this month’s meeting is the principle of social responsibility as set forth in the Universal Declaration of Bioethics and Human Rights of 2005, including article 14, which states that “the promotion of health and social development for their people is a central purpose of governments that all sectors of society share.” An IBC working group stressed that this implies that health should be one of the most important purposes of governments, while promoting health and social responsibility is an obligation shared by all societal actors, private and public. Source: UN News Service See also: Minority of UN Vote against Human Cloning CellNEWS - Tuesday, 08 March 2005 ......... ZenMaster

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ALS iPS Cells Created from Skin Cells of Human Lou Gehrig Sufferer

Research team creates human ALS motor neurons Thursday, 31 July 2008 A team of researchers from the Harvard Stem Cell Institute (HSCI) and Columbia University, in a collaboration catalyzed by the Project ALS/Jenifer Estess Laboratory for Stem Cell Research, has demonstrated that pluripotent stem cells generated from a patient with ALS (amyotrophic lateral sclerosis) can be directed to differentiate into motor neurons — the very brain cells destroyed by ALS. The results of the team's study appear in today's online issue of Science. This is the first published report to show that disease-specific stem cells may be derived from an individual patient. In the study, led by Kevin Eggan, of the Harvard Stem Cell Institute, skin cells taken from a patient with a familial form of ALS were induced to become pluripotent stem cells. Scientists then differentiated the pluripotent cells into motor neurons and glia (support cells in the brain) that featured an ALS genotype. "This is a seminal discovery," said Valerie Estess, director of research for Project ALS "The ability to derive ALS motor neurons through a simple skin biopsy opens the doors to improved drug discovery. For the first time, researchers will be able to look at ALS cells under a microscope and see why they die. If we can figure out how a person's motor neurons die, we will figure out how to save motor neurons." Starting in 1999, Project ALS recruited leading scientists and clinicians to define the potential role of stem cells in understanding and treating ALS, the fatal neurodegenerative disease, also known as Lou Gehrig's disease. Project ALS-funded scientists began by transplanting stem cells directly into mice with ALS, with limited success. More recent experiments have shown that stem cells may be more valuable as tools to understand the disease process and create mini-representations of disease — or assays — for the purpose of drug screening.

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Patient specific motor neurons created in the Eggan laboratory. Image courtesy of John Dimos/Eggan Lab at HSCI.

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"For the first time, we have the opportunity to examine cellular and molecular defects in motor neurons and glial cells derived from patients with ALS. And we can now begin drug screens on disease-specific classes of human motor neurons," said Thomas Jessell, a Howard Hughes Investigator at Columbia University, and Project ALS advisor. "Through the work of the Jenifer Estess Laboratory for Stem Cell Research we now can glimpse the new age of ALS research, an age of progress and promise." Co-author on the paper, Christopher Henderson, who is co-director of the Columbia University Center for Motor Neuron Biology and Disease, and senior scientific advisor to the Project ALS Laboratory, said: "It has been a privilege to collaborate with Kevin Eggan and his team and to contribute to this critical step forward. We will continue to work hand-in-hand with Harvard researchers and Project ALS to exploit the potential of these cells for drug screening". Three years ago, Project ALS asked Dr. Eggan, a stem cell expert, and Chris Henderson, Hynek Wichterle, as authorities on motor neuron biology and drug screening at Columbia University, to work together to understand ALS, one of our most complicated and devastating neurological disorders. Today's publication marks the first major breakthrough of this collaboration. About Project ALS: Project ALS is a non-profit 501©3 whose mission is to understand, treat, and cure ALS, also known as Lou Gehrig's disease. The hallmark of the company's approach is collaboration between researchers and clinicians, many of whom have not focused on ALS specifically, or worked together before. In ten years, Project ALS has raised over $37 million for research worldwide. Located in New York, the Project ALS/Jenifer Estess Laboratory for Stem Cell is the world's only privately funded laboratory to focus exclusively on stem cell and ALS. The laboratory was named for Project ALS founder Jenifer Estess, who died from ALS in 2003. See also: Neurons created from skin cells of elderly patients with ALS HSCI – July 31, 2008 Harvard-Columbia team creates neurons from ALS patient's skin cells Eurekalert! – July 31, 2008 NY Stem Cell Foundation plays critical funding role in major new ALS research announced today Eurekalert! – July 31, 2008 Induced Pluripotent Stem Cells Generated from Patients with ALS Can Be Differentiated into Motor Neurons John T. Dimos, Kit T. Rodolfa, Kathy K. Niakan, Laurin M. Weisenthal, Hiroshi Mitsumoto, Wendy Chung, Gist F. Croft, Genevieve Saphier, Rudy Leibel, Robin Goland, Hynek Wichterle, Christopher E. Henderson, Kevin Eggan Science July 31, 2008, DOI: 10.1126/science.1158799 ......... ZenMaster

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Study on Attitudes to Stem Cell Research

BBVA Foundation international study on attitudes to stem cell research Wednesday, 14 May 2008 Unlike most scientific and technological advances, which tend to take their place silently in society, biotechnology often finds itself the center of public debate and regulatory attention, due partly to the moral issues posed by many of its applications. In this second BBVA Foundation international study on “Attitudes to Biotechnology” (the first was in 2003), the sample has been enlarged from nine to twelve European countries (Austria, Czech Republic, Germany, Denmark, Spain, France, Ireland, Italy, Netherlands, Poland, United Kingdom and Sweden), with the addition of countries from other continents; namely the United States, Japan and Israel. The selection of countries was informed by both their demographical weight and their variability from the standpoint of religious beliefs and cultural traditions. Information was gathered through 1,500 face-to-face interviews in each country with subjects aged 18 and over (around 22,500 interviewees in all) conducted between April 2007 and February 2008. The design and analysis of the survey were the work of the Department of Social Studies and Public Opinion of the BBVA Foundation. The present study focuses on attitudes towards one biotechnology application: research with embryos for the purpose of obtaining stem cells. In particular, it analyzes how far public opinion is informed about stem cells, expectations and reservations regarding research with embryonic stem cells and differences in support for such research depending on the origin of the embryos used. Attention also goes to the attitudes held on the creation of hybrid embryos for stem cell research. PUBLIC UNDERSTANDING OF THE NATURE OF STEM CELLS The data show that the percentage of the population that admit having heard or read anything about this kind of cell was notably uneven across the survey countries: over 70% had heard or read about stem cells in Sweden and Denmark (86%), and also the United Kingdom, Netherlands and United States (between 70% and 75%); and over 55% in Italy, France, Ireland, Spain, the Czech Republic and Germany: while awareness of stem cells was less than 45% in Poland, Austria, Israel and Japan. As well as information about stem cells, the survey enquired about how far citizens understood the properties of such cells and the procedures used for obtaining them. The results point to a moderate understanding of stem cell properties: surpassing 50% in seven of the fifteen countries, between 40% and 50% in another four and below this threshold in the four remaining (Austria, Poland, Japan and Israel). In contrast, people had a poor understanding about how stem cells are extracted and the consequences for the embryo, with percentages no higher than 30% in the United States, between 15% and 20% in a further six countries and lower still in the remainder. VIEWS ABOUT RESEARCH WITH STEM CELLS In most societies there is a broad consensus around the usefulness of research with few-day-old human embryos in order to obtain stem cells. The mean agreement score with the idea that such research is very useful stood higher than the midpoint (5 on a scale from 0 to 10) in all countries except Austria, and was upwards of 6 points in nine of the fifteen countries, with Denmark and Sweden out in front. But this overall perception of usefulness does not rule out feelings of risk or moral dilemmas. Hence the data show considerable reservations about the risks entailed by researching with human embryos that are a few days old for the purpose of obtaining stem cells. There is general disagreement with the idea that this application poses no serious risks, with mean agreement scores below the midpoint (5) in eleven of the fifteen countries. The citizens perceiving least risk are the Danish and the Dutch, with Austrians, Americans and Japanese lined up at the other extreme. The moral or immoral nature of the application meets with divided opinions among survey countries. The majority view in Austria, Germany, Poland, Japan, Israel and United States is that this kind of research is immoral (mean agreement score above the midpoint on the scale), while those most strongly disagreeing with this supposed immorality are the citizens of Denmark, Spain, the United Kingdom and Italy (mean agreement score below the midpoint). Finally, opinions tend to cluster round the midpoint in the remainder of countries. POSSIBLE MEDICAL BENEFITS Debate and regulations regarding research with embryonic stem cells try to weigh up the medical benefits that may be obtained in future (the end pursued) against the moral reservations felt about this kind of research (the means utilized). When the possible medical benefits deriving from stem cell research are opposed in abstract terms to the rights of the embryo, opinions are divided both between and within countries:

• In Spain, the Czech Republic, Sweden, Denmark, France and the Netherlands, the balance leans to a greater or lesser extent towards the side of medical benefits. Hence the majority agree with the statement that “the medical benefits for many human beings that can perhaps be obtained in the future thanks to research with embryos that are a few days old are much more important than the embryos' rights”.
• In Austria, Ireland, Germany, Poland, the United States, Japan and Israel, the balance inclines more or less (depending on the country) towards the rights of embryos: that is, a majority dissent from the idea that “the medical benefits for many human beings that can perhaps be obtained in the future thanks to research with embryos that are a few days old are much more important than the embryos' rights”.
• Finally, the balance is more centered (mean value of 5) in the United Kingdom and Italy.

When the potential medical benefits are spelled out as treatments for what are seen as serious diseases (Parkinson's, Alzheimer's or diabetes), a majority in all countries declare themselves in favour of such research. The mean agreement with the assertion that “research with stem cells from embryos that are a few days old should be supported as a means of finding effective treatments for diseases such as Parkinson's, Alzheimer's or diabetes as soon as possible” was above the midpoint in every country with the exception of Austria, and exceeded 6 points in nine cases, with Spanish and Czech citizens agreeing most strongly. Besides moral objections, this kind of research meets with other reservations to do with ideas of what is natural or unnatural and concern about interfering with or altering the balance of nature. Citizens in most of the survey countries tended to agree that “research with human embryos that are a few days old is an unacceptable interference into the natural processes of life”, with agreement being firmest in Germany, Austria, Poland and Israel. There is also widespread concern that this kind of research may lead to other more dubious uses. The idea that “allowing research with embryos that are a few days old in order to obtain stem cells for use in medicine will open the door to other morally reprehensible uses” meets with considerable approval even in the countries favourably disposed to this application. The consensus round this view is especially marked in France, Germany and Japan. At the same time, research using embryonic stem cells touches on the moral or ethical framework of each individual, and in this sense moral criterion of religious inspiration is a key explanatory vector. In a context of plural opinions, the data show that the dominant view of the moral condition of the few-day-old human embryo is that it is close or identical to that of a human being. The strictly biological view finds widest support in Denmark and Sweden, where opinions are more equally distributed between those believing it makes no sense to talk about a moral condition of the embryo and those seeing it as close or identical to a human being. This view of the embryo as close or identical to a human is most frequently expressed in countries such as Austria, Germany and the United States. In Spain, opinions are quite sharply divided: 27% state that it makes no sense to talk about the moral condition of an embryo that is a few days old, while 25% take the intermediate position and another 35% see its moral condition as close or identical to that of a human being. ACCEPTANCE OF THE USE OF EMBRYOS DEPENDING ON THEIR ORIGIN Public debate and regulatory attention concerning research with stem cells has recently crystallized around two concrete scenarios: the use of spare embryos left over from fertility treatments and the use of embryos created specifically for biomedical research purposes. Citizens in most survey countries make differing judgments on these two scenarios, with acceptance of the use of spare embryos in all cases greater than that of embryos created for research. In the case of spare embryos, mean scores were in the approval zone in all countries except Austria (4.4) and Japan (4.6), and stood higher than 6 points in Denmark, Sweden, the Czech Republic, the Netherlands and Spain. In the case of embryos created for research, scores tended to range from 4 to 5 points, with support only at all emphatic in the Czech Republic (6.2). The citizens of Spain, Italy, the United Kingdom and Poland expressed marginal approval (just scraping in above 5 points on the scale) while remaining countries were all in the rejection zone. CREATION OF HYBRID EMBRYOS Faced with a shortage of human embryos for use in advancing stem cell research, British scientists have sought official permission to create hybrid embryos. In September 2007, the UK agency regulating embryo research and fertility treatments (Human Fertilization and Embryology Authority) approved the creation of hybrid embryos for the purpose of obtaining stem cells for biomedical research. The technique in question involves the implanting of the nucleus of an adult human cell into the egg of an animal from which the nucleus has been previously extracted. The BBVA Foundation survey also questioned citizens about their attitudes to such advances. The creation of hybrid embryos causes divided reactions both between and within countries. The baseline scenario meets with attitudes of rejection (below 5 on an acceptance scale from 0 to 10) in most of the countries studied. Only in the Czech Republic, Spain, Italy, Israel and Denmark does the mean score approach the midpoint on the scale. The citizens of Poland, France, Austria and Germany are the most critical of this application. Predominant in most countries is the fear that the technique could get out of control and lend itself to dangerous uses. This feeling appears to run deepest in Poland, France, Austria and Israel. A rather different reaction emerges in Denmark, the Netherlands and Sweden, where rejection of the creation of hybrids appears to have less to do with fear, and possibly more to do with perceptions that it is interfering with nature. .........

ZenMaster

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