1.   Do you envisage a requirement to take studies using human-animal chimera or hybrid embryos past the 14th day of development as per current HFEA regulations which prohibit the keeping or use of a human embryo which is more than 14 days old?

  No, I see no reason to undertake such studies. The intention of utilising cybrid-embryos is to generate embryonic stem cells, which will be harvested at blastocyst (Day 7 and Day 7+ of development, but not later than Day 9).

2.   Assuming that it is possible to produce cells eg embryonic stem cells created through somatic cell nuclear transfer of human genetic material into enucleated animal ova, do you envisage the need to establish totipotency or pluripotency of these cell lines? For what reasons might such studies be required?

  I believe it would be important to determine whether the cells were capable of establishing pluripotency. The ability to establish pluripotency is an indicator that these cells have been reprogrammed and are indeed embryonic stem cells that are capable of then differentiating into all cells of the body. I believe that by being able to apply criteria used in defining embryonic stem cells derived through in vitro fertilisation protocols would help in allowing these cells to be characterised as embryonic stem cells. This would validate their use to study what happens during the early stages of development in the disease state when lines of particular diseases have been established.

3.   What mechanisms would be used to establish totipotency or pluripotency of stem cell lines eg those produced through somatic cell nuclear transfer of human genetic material into enucleated animal ova? Is there a need for implantation of such cells into animals eg animal blastocysts?

  Such characterisation would involve those methods already established for analysis of embryonic stem cells generated from in vitro fertilised embryos, as described in the transcript. This would include assessment with biochemical markers, antibodies and RNA analysis to determine whether genes associated with pluripotency had been expressed. Markers associated with differentiation would also need to be tested as would those associated with trophectoderm. I do not see the need to generate chimeric animals for such a purpose.

4.   Do you envisage additional requirement for implantation of stem cell lines produced via somatic cell nuclear transfer of human genetic material into enucleated animal ova other than for establishment of pluripotency? For what reasons might such studies need to be conducted?

  No.

ADDITIONAL COMMENTS

  I am very much in favour of such work progressing forwards. I had contemplated submitting an application several months ago and had an initial informal discussion with the Human Fertilisation and Embryology Authority. I am now in the process of preparing such an application. My reason for wishing to use a cybrid-embryonic stem cell model is due to the obvious shortage of human oocytes from which such lines could be established. By generating cybrid-embryos and establishing embryonic stem cell lines, we will be able to develop disease models, especially those that are not currently available in any other form.

  My overriding research interest is in nucleo-mitochondrial interaction and how the nucleus would interact with another and similar populations of mitochondrial DNA. Consequently cybrid-embryos and embryonic stem cells would answer significant scientific questions in this respect. I also believe that this would help us understand some of the causes of mitochondrial DNA disease, where nucleo-mitochondrial interaction plays a significant role.

  I also feel that we should not necessarily view the generation of cybrid-embryonic stem cells with mixed populations of mitochondrial DNA as the final stage in development of such lineages. As part of my interest in understanding the transmission and replication of mitochondrial and nucleo-mitochondrial interaction, I feel that additional modifications will be required. It may be necessary to supplement oocytes with mitochondrial DNA from sources similar to the donor cell and these may need to be sources of oocyte mtDNA. It may also be necessary to deplete (partially or fully) the oocyte of its mitochondrial DNA in order that we generate functional and viable embryonic stem cells. This would be beneficial by ensuring that the cells would more likely be human-human cybrids and the proteins encoded by the genes of mitochondrial genome would interact appropriately with those of the nucleus that are responsible for generating cellular energy. These additional steps would enhance the efficiency and viability of such embryonic stem cells lineages.

February 2007