Supplementary submission from Dr Robin
Lovell Badge, Head of the Division of Developmental Genetics,
MRC National Institute for Medical Research
Please find below my responses to your additional
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?
1. For hybrid (or rather "cybrid")
embryos that involve the fusion of human somatic cells with enucleated
animal oocytes, I do not see any need to take them past the 14th
day. Indeed, I would expect that such embryos would begin to show
very compromised development beyond 14 days as it is around this
time that any mismatch between nuclear and mitochondrial genomes
is likely to become critical. While some cells may survive and
grow well, the embryo itself would not if there were too few cells
able to undergo the rapid cell divisions, cell movements and induction
events (where one tissue type influences another) that characterise
this phase of development. Moreover, as one of the major reasons
for making these cybrid embryos is to derive embryonic stem (ES)
cell lines, usually done with blastocysts at about day five, the
majority are likely to be destroyed well before the 14 day limit.
While it is possible that some scientists who are exploring the
mechanisms of reprogramming (ie how the somatic cell genome is
reprogrammed by the oocyte cytoplasm to behave like that of an
early embryonic cell) might want some embryos to develop further,
there are no compelling reasons to keep intact embryos beyond
14 days. Indeed, there are no currently available methods to do
so in vitro, and it would be impossible or pointless to try following
what happens to such embryos in utero. As no reputable scientist
wishes to carry out reproductive cloning and reprogramming is
being studied as a way to obtain useful cell types to study disease
and for therapies, it would anyway be far more appropriate to
test reprogramming in cells isolated from such embryos (as ES
cells, trophoblast stem cells, or other derivative cell types).
Methods to assay reprogramming at a molecular level are now very
good and will give more precise data than could be obtained by
following intact embryo development beyond 14 days. Transfer of
animal-human cybrid embryos into a woman's uterus is currently
illegal and I see no reason why this should change.
2. With respect to human-animal chimera
embryos, I also see no reason to take these beyond 14 days in
vitro (and as mentioned above there are no methods available to
this in a way that would preserve an intact embryo) or after transfer
into a women's uterus. There are occasions, however, where it
may be useful or desirable to allow development of certain types
of human-animal chimera beyond 14 days in an animal's uterus,
and even for the chimeras to be born.
3. As a general test of developmental potential
of a human stem cell line, including fetal or adult stem cells,
as well as ES cells. A common test of human ES cell potential
is to inject the cells into an ectopic site in a genetically immunocompromised
mouse (eg SCID mouse) where the cells are likely to form teratomas
or teratocarcinomas, tumours that contain many differentiated
cell types. Together with molecular analysis, this assay is sufficient
to say whether the cell line is really an ES cell line or not.
However, other types of stem cell do not give rise to teratomas,
but they may contribute to normal embryonic development after
introduction into preimplantation embryos, which are then transferred
to recipient females. For example, Catherine Verfaillie group
and Jonas Frisen's group carried out such a test with mouse mesenchymal
and neural stem cells respectively (Jiang et al (2002). Pluripotency
of mesenchymal stem cells derived from adult marrow. Nature 418:
41-9; Clarke et al (2000) Generalized potential of adult neural
stem cells. (2000) Science 288: 1660-3.)
4. There are some questions about this approach,
and it is very unlikely to be informative if human stem cells
are being tested after introduction into blastocysts of animal
species that are too distantly related in evolution to human and/or
whose rate of development is much faster than human. For example,
an attempt to derive chimeras after injection of human ES cells
into mouse blastocysts essentially failed because the human cells
were out-competed by the much faster dividing mouse cells (James,
Noggle, Swigut, Brivanlou (2006). Contribution of human embryonic
stem cells to mouse blastocysts. Dev Biol. 295 :90-102.) However,
such an experiment might work with the host embryos coming from
better matched animal species (sheep or cow for developmental
rate, or non-human primate). This might be considered by some
to be a better test of pluripotency than teratoma formation, as
one would be asking if the human stem cells can participate in
normal embryonic development. However, there is a "catch
22" here. We will only know if development is normal if someone
does the experiment and finds that it is.
5. One can also imagine this type of general
test being proposed as a way to screen for tumour forming potential,
with the idea being that tumours may only form from the stem cells
under test in a specific permissive tissue type or body location.
However, if the aim of the stem cell research is to obtain useful
cell types for therapy, then the assays for tumour formation will
need to be conducted anyway on the specific cells to be grafted
using standard assays in live born animals rather than embryos.
So I rather doubt this would be a significant use (eg a regulatory
requirement), but it should be borne in mind.
6. Rather than injecting the human stem
cells into preimplantation host embryos, it is also possible to
inject cells into postimplantation embryos already developing
in utero. For example, it is now technically possible using ultrasound
imaging to inject cells into defined parts of a mouse embryo from
about nine days of development (corresponding in stage to about
four weeks of human development). As techniques improve, it may
be possible to go even earlier or be even more precise about placing
the cells. The resulting embryos or live born offspring would
be classified as chimeras, but are likely to have the donor cells
in a specific tissue or organ. So far as I am aware, this has
so far been done only within the same animal species, eg mouse
neural cells into mouse embryo CNS, or mouse enteric (gut) nervous
system precursor cells into mouse embryonic guts. It can be done
as a test of developmental potential of the grafted cells or to
explore cell based therapies, and as such I can envisage the techniques
being used with human stem cells into animal embryos.
7. The same methods could also be used to
create "humanised" animal models, where specific animal
tissues are replaced by the equivalent human tissue. This is similar
to deriving mice that carry a human immune system or whose liver
is mostly made up of human hepatocytes, both of which are proving
to be extremely useful to explore infection with human-specific
pathogens or to test toxic substances or drug treatments, etc.
In these cases the human cells can be introduced postnatally as
the immune system and liver undergo significant regeneration,
but their introduction into embryos would allow a far greater
range of tissue types to be humanised. Obviously, replacing an
animal's entire CNS with human CNS would be considered contentious,
but replacing specific parts might not.
8. With respect to tests of developmental
potential, it is possible to obtain differentiation of ES cells
or other stem cells entirely in vitro to many cell types. If the
purpose of the research requires obtaining a specific cell type,
such as motor neurons, purely for in vitro study, then in vivo
studies may not always be necessary. However, it can be difficult
to obtain sufficient terminal differentiation in culture or appropriate
association with other cell types in order to test cell function.
So at some point human cells may have to be reintroduced into
an animal, although it would be best if this can be done in a
directed manner rather than simply by making general chimeras
at an early stage of development.
9. If one counts animal cells carrying one
or more human genes, or even a whole human chromosome as human-animal
hybrid cells, then this type of experiment has already been done.
The "Downs mouse" was made by fusing human somatic cells
with mouse ES cells, selecting ES cells that had retained an essentially
intact human chromosome 21, and then using these to make chimaeras
by injecting them into mouse blastocysts. These were then transferred
into recipient female mice and allowed to develop to give offspring.
These were then mated to other mice to establish a strain of mice
that carry human chromosome 21. These are proving to be a very
valuable model of Downs syndrome.
10. There are, therefore, a number of reasons
why human-animal chimeras might be required after 14 days of development,
but none of these would be capable of being successfully implanted
into a human uterus (which requires human trophectoderm), even
if this was legal, which it is not. Limits could be applied to
specified situations, eg only to the stage at which organs begin
to form might suffice for any general test of potential, or to
just prior to birth in some other cases, but it would be scientifically
inappropriate to apply these conditions to all types of chimera
and hybrid research. In my view each proposal to do research of
this type would have to be judged on its merits. They would all
fall under current Home Office regulation on animal experimentation,
which does include ethical review.
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?
11. It will be necessary to establish the
developmental potential of such cell lines. With respect to studies
addressing reprogramming, then tests of potential are essential
to verify that reprogramming to an embryonic state has occurred
and to ascertain how normal this is. For studies aimed at looking
at genetic disease, then it will be necessary to obtain the specific
cell type in which the disease is manifest (eg motor neurons)
and a general test of potency will be required to indicate whether
any particular cell line is likely to be useful. As noted above,
standard teratoma and in vitro differentiation tests should suffice
for most purposes. However, I think it is very difficult to exclude
the need to carry out chimera tests (of the sort mentioned above)
in some instances.
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?
12. Teratoma and in vitro differentiation
assays provide a reasonable test of pluripotency. Furthermore,
it may also be possible to develop purely molecular assays to
judge more precisely the developmental potential of a stem cell
line. We already know of a set of genes that must be active for
this to be the case, and microarrays and other more general screens
for gene expression, epigenetic state, etc, will allow better
predictions. But these predictions will be meaningless without
some ability to correlate the results of such assays with real
tests of potential. Again, differentiation in teratomas and in
vitro should generally suffice, but perhaps not in all instances.
I can envisage the need to explore how the human cells behave
after implantation into animalsindeed this is already done,
eg to test the ability of stem cells or their derivatives to contribute
in a functional way to, for example, the CNS in postnatal animals.
In my opinion, implanting human ES cells into animal blastocysts
is unlikely to be very useful as a test of pluripotency, especially
as it would require the use of blastocysts from closely matched
species to be meaningful. However, the introduction of cell types
derived from the ES cells into specific tissues in postimplantation
animal embryos may be very useful as a way to establish the potential
of these cells to contribute in a functional way to these tissues.
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?
13. As mentioned in my answer to question
1 above, it may be very useful to implant cells derived from human
ES cells into animal embryos, not into blastocysts, but at postimplantation
stages in order to create "humanised" animal models,
where disease processes and therapies can be tested.