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. 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 fertilization 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 1 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 center. 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 12 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 1 cell from a 4-cell stage embryo, allowing the remaining 3 cells to develop into a blastocyst (5-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 1 or 2 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 than 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 recognize their own cells as their own. By being able to select an HLA-identical embryo, the cord blood of the 'savior 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 1 cell at the 4-cell stage impairs the capacity of the embryo to develop into a healthy child in comparison with the removal of 1-2 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 1 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."
Illustration: Rendering of 8-cell stage of fertilized egg. --iStockphoto/Gary Caviness.
European Society of Human Reproduction & Embryology Press Release (07/09/08)
Science Daily (07/09/08)
Abstract (The 24th Annual Conf. of the European Society of Human Reproduction & Embryology; Session 69, O-274 Oral (07/09/08))