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Human embryonic stem cells developed from 4-cell embryo; world first may lessen ethical concerns

July 09, 2008

Barcelona, Spain: For the first time in the world scientists have succeeded in developing human embryonic stem cells (hESCs) from a single cell, or blastomere, of a 4-cell stage embryo, the 24th annual conference of the European Society of Human Reproduction and Embryology heard today (Wednesday 9 July). Dr. Hilde Van de Velde, from the Vrije Universiteit Brussel (VUB), Brussels, Belgium, said that their research meant that it might be possible in the future to produce hESC lines at an earlier stage without destroying the embryo.

Blastomeres are formed in the very early stages of embryonic development. About 24 hours after fertilisation the egg divides into two cells. Division into four cells occurs after 48 hours. After 96 hours, at the morula stage, the fertilized egg has divided four to five times. During this time the size of the embryo does not increase, so the cells become smaller and smaller and they are strongly attached to each other which makes them more difficult to manipulate. At this early stage important decisions are taken: inner cells will become the foetus (including germ cells) and outer cells will become trophoblast (the outermost layer of the embryo that attaches to the wall of the uterus and serves as a nutritive pathway). There was, until now, uncertainty about which stage of early development the blastomeres ceased to be totipotent, i.e. able to develop into all cell types of the body.

Worldwide, the majority of established hESC lines have been derived from the inner cells at the blastocyst stage; these are said to be pluripotent. "Previously, scientists have been able to derive hESC lines at the 8-cell stage," said Dr. Van de Velde, "but success rates were variable and it was necessary to culture them by mixing with established hESC lines. We have been able to derive hESCs at an earlier stage of embryonic development, and without the need for co-culture with established hESC lines. Now we have derived a second hESC from one cell of a 4-cell stage embryo. Given the complex nature of earlier attempts, we were pleased that we could develop a technique that seemed simple and was also reproducible."

The scientists used mature eggs donated by couples being treated at the University's IVF centre. Embryos were obtained after ICSI using sperm from a consenting donor. Three, good quality 4-cell stage embryos were split into 12 single blastomeres and allowed to grow in vitro to produce twelve morulas that were cultured in the conventional way for hESC derivation. From these twelve, one resulted in a stable hESC line. The scientists concluded that at least one cell was pluripotent.

These new data confirm their recent report that at the 4-cell stage the cells are equal and totipotent. "Now we will try to derive four hESC lines from the same embryo in order to compare the potency capacity of all four cells," said Dr. Van de Velde.

The work could have major ramifications for preimplantation genetic diagnosis (PGD), by enabling the biopsy of one cell from a 4-cell stage embryo, allowing the remaining three cells to develop into a blastocyst (five day embryo) which could be transferred into the uterus and develop into a healthy baby. "Currently, PGD is performed at the 8-cell stage, when one or two cells are removed; others have derived stable hESC lines at this stage but with low efficiency. If hESC derivation at the 4-cell stage turns out to be more efficient then at the 8-cell stage, we might consider to change our PGD policy in cases where we perform human leukocyte antigen (HLA) typing," said Dr. Van de Velde.

HLA molecules play an important role in the immune system by ensuring that our bodies recognise their own cells as their own. By being able to select an HLA-identical embryo, the cord blood of the 'saviour siblings' could help cure an older brother or sister suffering from a genetic disease affecting the production and/or function of hematopoietic (blood producing) stem cells. "We could also combine the pregnancy of an HLA-matched healthy baby with the derivation of a personal hESC line that could be used to generate in vitro hematopoietic stem cells as an additional source of hematopietic stem cells," she said.

"We need to determine whether the removal of one cell at the 4-cell stage impairs the capacity of the embryo to develop into a healthy child in comparison with the removal of one/two cells at the 8-cell stage. We understand that some people may have ethical concerns about the production of hESCs," said Dr. Van de Velde, "but we already know from cryopreserved embryos that the loss of one cell from a 4-cell stage embryo does not affect its capacity to implant in the womb. We believe that by making it possible to intervene at an earlier stage, and without destruction of the embryo, these ethical concerns will be diminished."

In fact, opinion among the public is very much in favour of stem cell research, another researcher told the conference. Ms Jaclyn Friedman, a clinical embryologist at Reproductive Biology Associates, Atlanta, Georgia, USA, undertook an on-line survey looking at attitudes towards IVF around the world. Two questions related specifically to human embryonic stem cell research and use in therapy.

"This is the first time an on-line questionnaire has been used to establish a cross-sectional view of ethical opinion in the global community," said Ms. Friedman. "Because stem cell research is increasingly important, we felt that we needed to gauge opinion among members of the public, as well as IVF patients, doctors and scientists working in the field."

The researchers analysed the following two questions:

"I believe that it is morally wrong to use embryonic stem cells for research"

and

"I believe that it is morally wrong to use embryonic stem cells for medical treatment."

573 people responded to these questions. The researchers found that 47.1% strongly disagreed with the viewpoint posed in question 1, whereas only 4.5% strongly agreed. 4.1% agreed, 10% were neutral, and 31.2% disagreed, meaning that in total 78.3% of respondents disagreed with the view that it is wrong to use hESCs in research.

For the second question, the results were broadly similar, although fewer people (3.1%) felt that it was morally wrong to use ESCs in medical treatment than in research. "We found no difference between male and female attitudes towards using hESCs for research, but when it came to medical treatment, men showed significantly more support than women. "We found no significant differences when we looked at particular age groups, but a higher level education of respondents correlated with greater support for the use of hESCs, both in research and medical treatment. There were no important differences among regions of the world.

"We hope that this questionnaire might act as a pilot study for something much larger and more representative of the views of the international community. But even with our existing numbers, we found enough difference between the views of people working in the field, the general public, and patients, to know that the results have statistical significance. Our study shows that public, patient, and scientific opinion is very much in favour of both stem cell research and the therapeutic use of stem cells in medical treatment. This is different from the perceived equal distribution for and against hESC use reported in the news media," she said.

The full results from the questionnaire will be available later this year on IVF.net.

European Society for Human Reproduction and Embryology