It will be necessary to test all human embryonic stem cell lines, those derived from both SCNT- and IVF-derived embryos, to ensure that the cells lines are pluripotent, that is capable of differentiating into cells of all three germ layers and thus into a wide range of cell types. The general methods for determining pluripotency are to:

  a)  allow embryonic stem cells to spontaneously differentiate or to use published differentiation protocols and then look for the generation of a number of different cell types across the three germ layers using markers that recognise specific cell types. The cells can thus be screened using immunocytochemistry against proteins expressed on various cells and tissue types or molecular teachniques (eg PCR) to look for the expression of very specific genes. This is the most standard and easiest test and one that every group uses.

  b)  to inject undifferentiated cells into the kidney capsule, testes or subcutaneously into mice lacking an immune system (SCIDs), where they will not be rejected by the mouse as foreign tissues. The hES cells will slowly differentiate into a wide variety of somatic stem cell populations, but will continue to grow as a disorganised mass of cells (referred to as teratomas which are benign tumours) representative of all three germ layers. Once the tumours are large enough to be felt, the mice are killed and the tumours are examined histologically for the presence of tissues representative of all three germ layers. The advantage of this test is that the differentiation patterns of the cells are more complex due to the influence of the in vivo environment and one observes more complex structured tissues such as glands, neural tube-like formation, gut and epithelium. This process however, is more expensive, time consuming and requires Home Office approval and licensing, but many researcher believe that this is a more rigorous test of pluripotency and should be required for all published cell lines.

  c)  inject labelled undifferentiated stem cells back into the environment from which they were derived, that is back into the inner cell mass of a developing blastocyst which is then implanted into a surrogate uterus. The resultant chimeric embryo can either be killed at mid-gestation when most of the organs have already been generated or the chimera could be allowed to proceed to term or adulthood. In either case, the chimera is then analysed for the presence of the labelled human cells in all major tissue types, such as the heart, brain, lungs, liver, bone marrow, skeletal muscle, and reproductive organs. If hES cells are truly pluripotent one would expect to find that the cells have been incorporated into all tissues, assuming that the gestational period is adequate for differentiation and integration to have occurred. To the best of my knowledge, this has only been performed in the US using mouse blastocysts. I was told confidentially that the mice has been killed on embryonic day 15 (normal gestation takes about 21 days) and the cells had not integrated into the mouse in a developmentally coherent fashion but had remained as an isolated clump of cells. There is also a published report (James et al., 2006, Devel Biol, 295, 90-102) where hES cells were injected into mouse blastocysts and the embryo were killed on gestation day eight which is very early in development and most of the tissues would not have formed. The majority of the embryo either did not contain cells or were developmentally abnormal. Only one of out 28 embryos appeared to be phenotypically normal and only had 10 visible human ES cells within the foregut and primordial brain.

  As you've already heard from Professor Austin Smith, very few of us in the scientific community think this last set of experiments are required or necessary to prove pluripotency. However, it could be that there are fundamental biological or therapeutic questions that might be answered in the future using this approach. I would recommend that the generation of chimeric embryos not be banned but rather regulated by the Home Office like all forms of animal experimentation. If and when applications to generate chimeric animals are received by the Home Office, I would recommend that specialist scientists and bioethicists be recruited and asked to provide peer review and a clear demonstration that this is necessary and important research before a license to generate chimera is granted.

February 2007