Select Committee on Science and Technology Written Evidence

Memorandum 61

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 questions:

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 animals—indeed 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.

February 2007

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