APPENDIX 42
Supplementary memorandum from Dr Neville
Cobbe, University of Edinburgh
CONCERNING THE FIRST APPLICATION TO CLONE
HUMAN EMBRYOS BY NUCLEAR TRANSFER IN THE UNITED KINGDOM
INTRODUCTION
My attention has recently been drawn to an application
by Professor Alison Murdoch to generate stem cells from embryos
produced by parthenogenetic activation or cloning by nuclear transfer.
I am keenly aware that the proposed research has provoked public
outcry and I have therefore not bothered to trouble the HFEA with
additional objections, preferring instead to stand back for now
and see how the application will be handled. Unfortunately, I
do not have access to Professor Alison Murdoch's full application
so I am unaware of how she has attempted to justify the proposed
research or account for its feasibility. However, I would like
to draw your attention to several concerns based primarily on
information available in the lay summary 4 and various media reports
or press releases. I trust the information below will be of interest
to you in evaluating any future decision made by the HFEA on this
matter.
AN EMBRYO
FROM AN
EGG
It is suggested in the lay summary for Professor
Alison Murdoch's application that the derivation of stem cells
from embryos produced by parthenogenetic activation is intended
to overcome rejection of genetically dissimilar transplanted cells
by recipients. Naturally, if any suitable stem cell lines could
be generated in this way, then their therapeutic use would be
restricted to the treatment of pre-menopausal women from whom
eggs were obtained. However, research spanning over two decades
has repeatedly shown that parthenotes in various mammalian species
display only limited developmental competence due to the need
for differential maternal and paternal genomic imprinting in both
the embryo and extraembryonic tissues. Given this lack of developmental
competence, it could be argued that parthenotes might offer a
more ethically acceptable means to generate embryonic stem cells
without needing to create or destroy a potentially viable embryo.
However, it would also appear that the therapeutic potential of
parthenote derived stem cells may be limited by their reduced
developmental competence. In addition, it has been pointed out
that parthenogenetic cell lines may be of limited use in therapies
involving tissue reconstitution because such cells would be at
a competitive growth disadvantage. Moreover, even though the derivation
of pluripotent cell lines from parthenogenetically activated cynomolgus
macaque eggs has recently been described, the developmental competence
of these cells was only demonstrated by their ability to form
teratomas following transplantation, just as the spontaneous development
of unfertilized eggs in women usually gives rise to ovarian teratomas.
Consequently, whilst the production of cell
lines from parthenogenetically activated eggs does not pose many
of the same ethical concerns as research necessitating the deliberate
destruction of potentially viable human embryos, it is still far
from clear whether such cells would be either useful or safe in
a therapeutic context. Whilst it may be possible to support limited
use of parthenote-derived stem cells for research purposes, I
would hope that no prior clinical use would be made of such cells
without thoroughly evaluating the consequences of transplantation
in rodent models. I would also question the motivation for subjecting
women to aggressive ovarian stimulation regimens if their eggs
are then to be used in research that shows so little promise of
generating useful stem cells. Referring to the egg donors, I note
that Professor Murdoch has said: "Although the studies will
not directly help them, they are playing a vital role in helping
other patients". This would be a truly bizarre comment to
make regarding the creation of parthenotes, given that only the
women who donate these eggs would be compatible matches for any
resultant stem cells.
THE CASE
FOR CLONING?
I am however, more concerned by the other proposal
by Professor Alison Murdoch to derive stem cell lines by nuclear
transfer from adult epithelial cells to enucleated eggs. Unlike
the production of cell lines from parthenotes, this research necessarily
involves the deliberate destruction of potentially viable embryos.
Furthermore, there is already concern that advancing the cause
of "therapeutic" cloning research is simply providing
the means to make human reproductive cloning a reality. Whilst
the latter is currently illegal in the United Kingdom, I would
have thought a moratorium on human cloning research would be wise
until an international ban on reproductive cloning can be achieved.
Aside from the obvious ethical concerns, it is unclear to me what
Professor Alison Murdoch hopes to accomplish that will not simply
be a repetition of work performed in South Korea. Although not
explicitly stated in the Lay Summary, it appears that the cell
lines are intended for use in developing new treatments for diabetes,
raising the question of how the resulting cells might advance
previous work performed in Israel. Indeed, as the lay summary
concludes with a statement indicating that this work is of a highly
preliminary nature, it is worth questioning why it must be performed
with human embryos rather than with embryos from various model
organism species.
It should be remembered that type I diabetes
mellitus is typically caused by autoimmune destruction of the
insulin-producing ß-cells in pancreatic islets. Of course,
given that the stem cell lines that Professor Alison Murdoch and
Dr Miodrag Stojkovic intend to create would be genetically identical
to the diabetic patients providing initial tissue samples, would
not any resulting ß-cells be just as prone to destruction
by the body's own immune system after transplantation? Indeed,
even Professor Ian Wilmut has recently commented that immunologically
identical cells are expected to induce the same rejection in patients
suffering from autoimmune diseases such as type 1 diabetes. If,
however, it is insisted that transplanted pancreatic material
must be an autograft, then I would question the reluctance of
the Newcastle research team to investigate the potential of either
pluripotent stem cells from adult sources or dedifferentiated
cells treated with compounds related to reversine to generate
stem cell like progenitors. Can anyone categorically deny the
possibility that islets or ß-cells suitable for transplantation
could be produced from adult cells? Has it is also been demonstrated
that gene therapy on adult cells would fail to produce glucose-responsive
and insulin producing cells suitable for transplantation?
ACHIEVEMENTS WITH
ADULT CELLS
On the contrary, it appears that insulin-producing
cells with the ability to reverse hyperglycaemia in diabetic mice
may be derived in vitro from adult hepatic stem cells.
Similarly, adult bone marrow-derived cells have been shown to
be capable of trans-differentiating into insulin-producing pancreatic
cells in vitro, which normalise glycaemia after transplantation
into diabetic mice. The derivation of insulin secreting and glucose-competent
endocrine cells from bone marrow has also been demonstrated in
vivo in non-diabetic mice, without evidence of cell fusion.
Furthermore, transplanted bone marrow-derived stem cells may alternatively
reverse hyperglycemia caused by drug induced pancreatic damage
by giving rise to mature donor endothelial cells, which in turn
somehow facilitate pancreatic regeneration. Although other groups
have failed to produce similar results, this may be due to the
introduction of bone marrow-derived cells prior to inflicting
pancreatic injury, rather than as a subsequent therapeutic intervention,
since the frequency of insulin-positive donor cells has been shown
to increase dramatically after pancreatic damage. Strikingly,
following transplantation of splenocytes into severely diabetic
autoimmune mice and restoration of normoglycaemia, it was found
that between 29% and 79% of islet cells were of donor origin (without
evidence of significant cell fusion), whilst no islets solely
of host origin were detected.
Although various previous studies have proposed
the existence of endogenous pancreatic stem cells, it has recently
been shown that most pancreatic regeneration in mice results from
self-duplication of pre-existing ß-cells. Given this regenerative
capacity, it has been suggested that therapies to reverse autoimmune
diabetes need not necessarily incorporate transplantation of exogenous
adult islets but may also be achieved by re-educating naïve
T cells through presentation of matched MHC class I molecules
and self antigens. For example, treatment of diabetic mice with
an inducer of TNF-α production can eliminate autoreactive
lymphocytes, permitting the reappearance of endogenous host ß-cell
function following islet transplantation. Furthermore, endogenous
pancreatic cells are able to give rise to new islets following
treatment with either irradiated splenocytes or spleen cell fractions
positive for the leukocyte common antigen (albeit less efficiently
than when non-lymphoid donor cells directly contribute to islet
regeneration). Indeed, it appears that endogenous ß-cell
function can be restored to physiologically sufficient levels
with as little as 1% donor chimerism in peripheral blood leukocytes
following allogeneic bone marrow transplantation in overtly diabetic
mice. In conclusion, it appears that pancreatic ß-cell function
can be restored using adult cells in a variety of ways, depending
on the cause and severity of diabetes in each case.
Aside from the use of stem cells differentiated
into ß-cells, gene therapy with other cell lines also offers
a promising route to long-term therapeutic treatment of diabetes.
Recently, a reversibly immortalized human hepatocyte line expressing
a single-chain modified insulin under the transcriptional control
of a glucose-sensitive promoter was shown to significantly prolong
the survival and lower blood glucose of diabetic pigs previously
undergoing total pancreatectomy, without evidence of tumour formation.
It is also expected that autografts of insulin-producing hepatocytes
would avoid autoimmune attack, since previous studies showed that
endogenous hepatocytes expressing transgenic insulin were not
attacked by the autoimmune response in NOD mice. This is probably
because the autoimmune response is specific to ß-cell antigens,
whilst transgenic hepatocytes still express various liver genes.
Alternatively, expression of insulin in response to glucose may
be activated by introduction of a Pdx1 transgene into hepatocyes
or their progenitors, which has previously been shown to replace
ß-cell function in vivo. Based on previous studies
in mice, it is seems likely that Pax4 overexpression in hepatocytes
may also normalise glycaemia by promoting even greater insulin
expression, but without the same risk of tumorigenesis associated
with embryonic stem cells.
EMBRYONIC STEM
CELLS
By contrast, the general protocol used to derive
insulin-positive cells from embryonic stem cells by enriching
for nestin-positive cells has been shown to select for cells with
mostly neuronal features and not ß-cells. Although insulin-positive
cells have been isolated using this procedure, it has since been
shown that these are commonly a consequence of insulin uptake
from the culture medium, rather than differentiation into actual
ß-cells. In addition, such cells are not suitable for transplantation
into diabetic patients due to the formation of teratomas and their
failure to stably correct hyperglycaemia. Moreover, although nestin-positive
cells can contribute to the vasculature of the islets, they do
not contribute to endocrine cells or other cells in the pancreas,
whilst insulin-positive cells have been shown to exist in the
liver, adipose tissue, spleen, bone marrow and thymus of different
diabetic mouse and rat models. Consequently, although the treatment
of neuronal-like nestin-positive cells with an inhibitor of phosphoinositide
3-kinase appears to further enrich for insulin-expressing cells,
it is unlikely that these cells are true ß-cells as they
remain nestin-positive, appear to lack pancreatic polypeptide
or somatostatin and display intracellular insulin levels only
10% of those in isolated pancreatic islets, with more proinsulin
accumulation than in ß-cells. Even with these apparently
differentiated cells, the risk of tumorigenesis still cannot be
excluded. Similarly, although the expression of a Pax4 transgene
in embryonic stem cells resulted in up to 80% of embryonic stem
cells developing into insulin-producing cells, it was nevertheless
acknowledged that the remaining undifferentiated cells could still
be oncogenic.
The grave risks currently associated with any
therapeutic use of embryonic stem cells and in particular those
derived from nuclear transfer should make most people cautious
of their use in patient treatments. Such risks are acknowledged
even by proponents of research involving human embryos and are
referred to in Recital 12 of the EU human tissue directive. By
contrast, it appears Dr. Miodrag Stojkovic just intends "to
bring faster and more efficiently stem cells from bench to patient's
bed". It also remains to be demonstrated whether or not recently
described insulin-producing clusters derived from human embryonic
stem cells are likely to form teratomas following allograft transplantation,
though this seems highly probable given that the same differentiation
protocol fails to prevent the formation of teratomas in mice.
Personally, I would have thought that further study of differentiation
protocols and transplantation in model organisms would be warranted
before experimenting on human subjects, in keeping with the Nuremberg
Code and the Declaration of Helsinki.
OBTAINING OVA
Professor Ian Wilmut has himself acknowledged
that "therapeutic cloning is unlikely to be practical for
routine use", since the process would require an inordinate
supply of eggs. The recently published South Korean study used
a total of 242 fresh oocytes donated by healthy women, from which
only one embryonic stem cell line was derived. Whilst it may be
possible to improve the efficiency of nuclear transfer in the
future, it should be recognised that the perfection of such techniques
and their inevitable publication would simply facilitate reproductive
cloning of humans by mavericks elsewhere. I gather that Professor
Alison Murdoch intends to use the spare 30% or so eggs produced
during IVF treatments that might otherwise be discarded. However,
if the eggs to be used for cloning by nuclear transfer are inferior
and incapable of being fertilised in IVF, then the chances of
even producing blastocysts would appear to be greatly reduced,
never mind generating useful stem cells. If it is argued that
any resultant embryos would have such epigenetic defects as to
render them inviable, then these very same features would surely
limit the developmental potential of any resulting stem cells.
Moreover, the increased chance of epigenetic dysregulation would
probably lead to further risks of tumorigenesis after implantation.
On the other hand, if embryonic cell lines are to be used to study
the aetiology of disease progression during development (which
itself raises concerns about whether or not the current 14 day
limit on embryo experimentation might be breached), then many
cell lines might be required in order to control for additional
variation due to epigenetic effects.
Even if a vast excess of eggs left over from
IVF treatment might be available for research purposes, it is
then worth questioning why such an excess must exist to begin
with. One might assume that vast numbers of eggs are required
to generate enough embryos for successful implantation. However,
it should be noted that pregnancy rates over 30% have been reported
after transfer of single embryos at the cleavage stage, whilst
pregnancy rates in excess of 50% have also been described following
single blastocyst transfer (Note that variation in the highest
success rates may be explained by differences in patient populations,
since the merits of blastocyst versus cleavage stage embryo transfer
appear debatable). The rationale for creating excess human embryos
for freezing is also questionable, given that cryopreservation
has been shown to be associated with blastomere loss and reduced
implantation potential with around a quarter of embryos failing
to survive thawing. Since the production of excess embryos does
not necessarily improve pregnancy outcomes and increases the risk
of multiple pregnancy if more than one embryo is transferred,
this raises serious questions regarding why so many eggs must
be retrieved from female patients, especially given the elevated
risks of ovarian hyperstimulation syndrome following aggressive
hormonal treatments.
CONCLUSION
Whereas the Warnock Committee previously concluded
that the early embryo has at least a "special status"
and "should be afforded some protection in law", Professor
Alison Murdoch has been quoted as saying that "embryos have
no more moral status than blood taken from a patient". Therefore,
I can only conclude that the principal reason for insisting on
such research is simply a consequence of easy access to materials
for which there is little or no respect, rather than a desire
to pursue the most promising research avenues in the search for
effective therapies. It had been my previous understanding that
the HFEA's research licensing committee would only approve applications
for research on human embryos if researchers could show they are
not using excessive numbers of embryos and there is no other way
of doing the research. It stretches one's imagination trying to
conceive how this could be the case with Professor Alison Murdoch's
application, so I would fully expect the HFEA to reject such a
request. In stark contrast to any claim that preventing therapeutic
cloning or embryonic stem cell research would unfairly discriminate
against diabetics, it is my impression after surveying the research
literature to date that the real threat to finding cures is the
diversion of funding and expertise into such apparently irresponsible
research.
I hope these comments will be useful to the
Science and Technology Select Committee as they consider the ramifications
of such research in relation to revision of the 1990 HFE Act.
As ever, I am willing to try to provide reference material should
any Committee members wish to examine the research literature
for themselves. I would also encourage the Committee to independently
and objectively evaluate both the feasibility of Professor Alison
Murdoch's proposals and the wisdom of the HFEA in its eventual
handling of her application.
June 2004
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