Supplementary submission from Dr Jus St
John, University of Birmingham
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
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?
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.