Until now, scientists investigating human cloning for therapeutic purposes have been limited to using mature eggs (oocytes) that have reached the metaphase II stage (MII) at which ovulation and fertilisation occurs in humans. However, there are few human MII oocytes available for research because almost all that are retrieved from women seeking fertility treatment are used to treat the patient. Immature oocytes are not used routinely for treatment at present, and so any that are retrieved can be donated for research. These immature oocytes are arrested in the prophase I stage, before meiotic division is complete, when the enlarged nucleus is called the germinal vesicle (GV).
Bjorn Heindryckx, a PhD student at the Infertility Centre at Ghent University Hospital, Belgium, and his colleagues, matured GV oocytes in culture in the laboratory for 44 hours, after which time 85% of the GV oocytes had developed into MII oocytes. From each of these, they removed the nuclear apparatus, which contained the chromosomes that held all the genetic information. Using conventional ICSI techniques, they injected into the empty oocytes the nuclei taken from somatic cumulus1 cells (i.e. non-germ cells) of another person – a process known as non-autologous nuclear transfer. After time for nuclear re-programming the oocytes were artificially activated by incubation in a medium containing calcium ionophore, which enabled the injected nucleus to prepare for the first embryonic division.
Mr Heindryckx said: "Eighteen out of the 25 in vitro matured MII oocytes survived this nuclear transfer. Of these, 11 showed the formation of one pronucleus. In normal fertilisation the formation of male and female pronuclei is an important stage just before the maternal and paternal chromosomes start to pair up in preparation for the first cell division. In the case of these oocytes they had undergone a pseudo-fertilisation because the pronuclei were derived from whole foreign nuclei, each one with a complete set of homologous [matching] chromosomes, instead of from sperm and eggs which carry only one set of chromosomes each."
Five oocytes divided to the two-cell stage, and of these, three continued to divide to the six- to ten-cell stage. One embryo continued to develop to the compacted stage, when the individual cells started to flatten and increase their contact with one another.
Mr Heindryckx said: "To our knowledge, this is the first report describing the development of cloned human embryos using in vitro matured oocytes and non-autologous transfer via a conventional method of nuclear transfer.
"Our final goal is to use human therapeutic cloning for infertility treatment by creating artificial eggs and sperm for patients who are infertile because of absence or premature loss of eggs or sperm. We would do this by isolating embryonic stem cell lines from cloned early embryos and driving these embryonic stem cells to develop into eggs and sperm in the laboratory."
However, Mr Heindryckx warned that there was a long way to go and many problems to overcome before he and his colleagues could reach their goal.
"None of these early embryos developed to the blastocyst stage, and failure to do so could reveal some problems in gene activation, especially in cloned embryos. So, first we have to understand how to get cloned blastocysts of good quality from in vitro matured oocytes. This will be difficult because it is well known that embryonic development is compromised when in vitro matured oocytes are used, and in cloning technology the oocytes undergo intensive micromanipulation, which makes it even harder to develop good quality blastocysts. Once we have achieved this, the next step will be the isolation of the Inner Cell Mass (ICM) from which we can obtain embryonic stem cells.
"The availability of human oocytes is a major obstacle at the moment for research into therapeutic cloning. Therefore, we consider this research is important because it makes best use of more easily available biological material – in this case, immature oocytes."
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