The 'Infertility Treatment Act' 1995, does not include a definition of embryonic stem cells. The AHEC report (Scientific, Ethical and Regulatory Considerations relevant to cloning of Human beings, 16 December 1998) defines the embryonic stem cell as an "undifferentiated cell, which is a precursor to a number of differentiated cell types."(page 51). For the purposes of the Infertility treatment Act 1995 ES cells are neither gametes nor embryos. Therefore, they are not within the requirements related to research, nor within the approval processes, in relation to import and export of gametes and embryos, prescribed in section 56. The Authority, therefore, "has no statutory power under the Infertility Treatment Act 1995 to prescribe certain actions or requirements in relation to the importation of ES cells into Victoria or in relation to their use in research in Victoria."1
Recently, the hypothesis has been put forward, that the cells of the embryo are all stem cells, which can be made to grow indefinitely at the embryonic stem cell stage, and later changed to any cell in the body, and induced to form organs, and supply an inexhaustible supply of replacement cells or organs.
A close examination of the literature, shows that embryonal cells are preprogrammed to develop as a normal embryo develops, and far from directing change, the researchers have merely grown what parts of the embryo they could, and when a recognisable tissue had formed, claimed that they had directed differentiation.
The actual cells, which they identify as embryonic stem cells, keep differentiating in so many different ways, that they produce only teratomas, i.e. masses of mixed adult tissues, and are quite unsuitable for use for transplantation.
Sir Arthur Keith published the first of his treatises on "Human Embryology and morphology" in 1902.
In 1933, a group of American Embryologists, under the aegis of the Carnegie Institution of Washington, began to maintain a colony of Rhesus monkeys, and studied every stage passed through by the developing embryo of that animal from the first to the 35th day.
These stages, and the rate at which they were passed through, was very similar to those of the human embryo -- at least for the first 5 weeks.2
Gradually more knowledge of human development accumulated, as embryos of different ages were found.
Of recent years, the work of Professor Axel Ingelman-Sundberg of the Women's Clinic at the Sabbatsberg Hospital and many others, has been made accessible to the world at large by the photography of Lennart Nilsson.3 4
We now know, that in the developing human embryo, primitive nerve cells are visible and growing at three weeks.5
From the moment of successful sperm penetration, an organised train of events has been set in place. "If all the cells were alike, they would probably divide simultaneously; there would be a 4-cell stage, an 8-cell, a 16 cell, etc. But stages with an odd number of cells have been observed. This implies that some cells divide faster than others. From a single cell, the fertilized ovum, there have emerged several kinds of cells."6... Although all cells have the same chromosomes, they are only using that part of the genetic structure necessary for them at that time. Throughout development, "...this selection prevails, resulting not in millions of copies of the ovum, but in a highly organized cell community in which every part has been given its special task."7 In other words, they are already differentiating, following a master plan in which they play particular roles.
This is seen in Nilsson's beautiful photograph of a four day blastocyst.8
There is a clear area of cell products, or cleft, centrally. The embryo proper (below) faces this cleft. In the centre of the embryo is a mass of the tightly packed, rapidly growing, smaller cells. These are roundish or triangular or smaller and bead-like, all different kinds.
Anteriorly we can see different large, round and globular cells to the front of the embryo, with some spreading or spread around the cleft.
Posteriorly, the embryo abuts the cyst wall, and here the cells have become larger, flatter, and more indistinct, with a narrow dark space just appearing between them and the blastocyst wall.
This space will enlarge forming the amniotic cavity and enclosing the embryo in its self-formed fluid-filled shock absorbent envelope.
Further along, smaller squarish and rectangular cells approximate the blastocyst wall. The anterior cells will be endoderm, and we know will continue to line the cleft and form the yolk sac, which in turn will form primitive blood cells and primitive gut and urogenital tracts.
The most posterior cells will form the trophoblastic wall, which will become the chorion and placenta. Those cells abutting these, will form the back of the embryo, the brain and spinal cord, and skin. In between, in the middle, will develop blood and Iymph vessels, blood cells, heart muscle, liver, ovaries, testicles and kidneys.
In the mean time, back to the blastocyst, where with furious energy, the cells continue to multiply and differentiate.
At day 7 or 8, the embryo bursts free from the blastocyst shell provided originally by the zone pellucida of the ovum.9
Hitherto it has been a contained, evolving entity, producing some hormones and sugars, but now it is ready to settle into the maternal endometrium and form a placenta for sustenance and exchange.
"A careful examination of the blastocyst's surface reveals that almost every cell is unlike another. Some have long projections, others have short ones and some lack projections altogether. This is called cell differentiation."10
Cells differentiate according to internal and external cues.
The external cues come from the region in which the cell finds itself, and the milieu of molecules, proteins, and chemicals formed by it and surrounding and supporting cells.
The internal cues are given by its own genes and the proteins they express, some 30,000 to 100,000.
The whole organised, living, developing being is the human individual who commenced life with the entry of the father's sperm into the mother's ovum.
In April 2000, an article entitled "Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro" was published by Reubinoff et al.11
The local Melbourne press reported that embryonic stem cells were cells derived from the early embryo, which have the potential to become any cell in the body, and could be kept alive indefinitely.
The Age said that the Monash group had confirmed that stem cells can be grown from human embryos. Professor Trounson said that it was now possible to have a renewable supply of ES cells without having to use more human embryos; they had gone further, by being able to get the embryonic stem cells to grow into nerve cells in the laboratory, in a controlled manner.12
To test these claims, we turn to the original article:
It claims far less. It states "Because no diploid human pluripotential stem cell has thus far been cloned, there is a remote possibility that more than one cell type is present in the cultures and may account for the variety of differentiated cells observed in vitro and in vivo."
When one reads the experimental protocol -- "The outer trophectoderm layer was removed from four blastocysts by immunosurgery to isolate inner cell masses (ICM), which were then plated onto a feeder layer of mouse embryo fibroblasts"13 and we realise that the entire embryo, minus only the placental and membrane progenitors, was plated; this is a very likely, not a remote, possibility. There is a picture of a largish clump of cells on a bed of fibroblasts, and we are told that growth from small clumps of cells (<10cells) was not possible.
Within several days, groups of small, tightly packed cells had begun to proliferate from 2 of the 4 embryo cell masses, and surrounding them were outgrowths of differentiated cells.
Two of the embryos were trying desperately to cling to life, and do what they were programmed to do. Both these were found to be female. The other two had presumably succumbed.
The growing small cells were then mechanically dissociated from their more differentiated cells, and following replating, they gave rise to flat colonies of cells which, the researchers decided, looked like human embryonal carcinoma cells, or primate embryonic stem cells. These were divided into further clumps, but along the way, cells looking like endoderm appeared. In this way, Embryo HES1 grew for 64 passages in vitro, and HES2 for 44 passages.
Both so-called embryo cell lines, when put under standard culture conditions, underwent spontaneous differentiation, and considerable cell death. Death was apparently delayed by nutrition from the mouse fibroblast feeder layer. But with prolonged cultivation of 4-7 weeks, multicellular differentiation, including to nerve and muscle, was observed.
Early stages of neuronal differentiation were first detected in cultures grown for approximately three weeks. From our knowledge of human embryology, we are not surprised at this. It is normal in an embryo of that age.
Claims that these parts of haphazard embryo growth represent derivation from immortal stem cells, which ean be farmed indefinitely, are not proven.
In a second article, "Neural Progenitors from human embryonic stem cells,"14 we find that:
"To derive enriched preparations of neural progenitors, differentiation of human ES cells was induced by prolonged culture (3 to 4 weeks) without replacing of the mouse embryonic fibroblast feeder layer."
During the next 2 weeks of culture, distinct areas of small tightly packed cells with a uniformly white-gray and opaque appearance under dark field stereomicroscopy, and surrounded by diverse liver, cardiac, and muscle reacting cells, were identified. Clumps of about 150 of these cells were mechanically isolated and replated in serum free medium where they formed "free floating spheres."
So to get their neurones, they just grew embryonic tissue undisturbed until it became obviously nervous tissue, and then dissected it out.
The spheres grew rapidly in the first 5-6 weeks, less so for 18-22 weeks, after which the spheres ceased to enlarge, or became smaller.
Hardly a recipe for immortality, or large scale production.
In this time, these immature nerve cells became more mature. Not surprising, when we consider that a twenty-two week foetus can be born alive, and in rare cases survive to adulthood. It is also not surprising that they failed to differentiate as well as in a healthy, undisturbed human embryo-foetus.
But what is interesting, is that in an apparently pure culture of nerve cells, the expression of transcripts of non-neural markers was evident after prolonged cultivation of the spheres. As the paper reports, it could represent contamination by a small number of non-neural cells during derivation of the cultures, or plasticity of primitive neural progenitors that expressed marker of, or gave rise to cells from, other lineages.
The in vivo parts of the experiments are worth looking at.
In the first paper, when cells of HES1 and HES2, of either early or late passage, were innoculated beneath the testis capsule of severe combined immunodeficient (so that they would not reject them) mice, all mice developed tumours. These were teratomas that contained tissues of all three germ layers including cartilage, squamous epithelium, primitive neuroectoderm, ganglionic structures, muscle, bone, and glandular epithelium. Embryonal carcinoma was not seen in any lesion.
In the second paper, the isolated immature nerve cells were implanted into the lateral ventricles of newborn mice. Cells survived in 9 out of 14 recipients.
One week after transplantation, clusters of donor cells lined the ventricular wall. At 4-6 weeks, human cells had left the ventricles and migrated in large numbers mainly as individual cells into the host brain parenchyma and were widely distributed. Some location specific differentiation was observed -- neurones in the olfactory bulb where neurogenesis occurs after birth, and gliogenesis in the white matter.
There was no evidence that they would behave in the same way in adult mice, let alone humans.
However they did adduce evidence that these transplanted cells were NOT stem cells, but in the main, neural progenitor cells, which had been farmed from the embryo. Their staining methods did show instability in the physiology of these apparently similar cells, warning of possible dangerous reversion to stem cell status, or contamination with the same stem cells.
This is important when one considers that in the USA, a man with Parkinson's Disease, who was given an infusion of fetal brain cells into his ventricles, died suddenly two years later. At postmortem, his cerebral ventricles were found to be choked with teratoma, which had stopped his breathing.15 Mice have a life expectancy of two years, so there is possibly time for teratoma formation in mice to take place in two years, and no guarantee that it is not going to happen in humans at a later date.
What we have learnt from these two experiments is,
It defies belief, that these two papers are cited as reasons for dismantling all the protection we have endeavoured to set up for our infertile and embryonic patients.
Further consideration needs to be given to the fact that on May 4th 2002, at the AGM of the Medico-Legal Society of Victoria, Alan Trounson produced a slide of what looked like a 4.4 day mouse embryo, and announced that you could get neurones by withdrawing cells from the head region, and cardiac muscle cells from the cardiac region.
Comparing the gestation period of a mouse 19-21 days, with that of a human 38 weeks, an equivalent human embryo would be about 4-5 weeks old. Nerve and muscle cells are developed then.
The Age (June 4,2002) reported that a cow's kidney had been cloned by scientists from Advanced Cell Technology in Massachusetts and the Children's Hospital in Boston, from an 8 week old cow's foetus, equivalent to a 7.75 weeks human foetus (cows' gestation period is 273 days, humans' is 266 days).
Do our scientists propose to grow human embryos to 4-5 weeks, and 7-8 weeks respectively, to farm human cells and organs'?
Disorganising the embryo in attempts to find stem cells, defeats the aims of replacement, and underlines the futility of such research.
This provides a powerful argument for heeding the ethicists.
What is also worrying, is that although a synchrotron which produces a powerful beam of light and is believed to be better than an electron microscope, could be a powerful aid to science, there is no mechanism so far proposed for access to its use for more laudable projects.
1 Presentation to Victorian Division of Doctors Who Respect Life by Helen Szote, Chief Executive Officer of Infertility Treatment Authority, Victoria 17.11 2001. [Back]
2 Human Embryology and Morphology. Sir Arthur Keith 6th Edition. 1948. [Back]
3 The Everyday Miracle Lennart Nilsson, Axel Ingelman-Sundberg 1965 Stockholm Sweden [Back]
4 A Child is Born Lennart Nilsson & Lars Hamberger 1990 Dell Publishing [Back]
5 A Child is Born Lennart Nilsson 1990: Bantam Doubleday Dell Publishing Group Inc. p 77 [Back]
6 The Everyday Miracle Lennart Nilsson, Axel Ingelman-Sundberg, Claes Wirsen p 40 [Back]
7 The Everyday Miracle p 40 [Back]
8 A Child is Born p 63, main picture [Back]
9 A Child is Born p 63, lower right-hand corner picture [Back]
10 A Child is Born p 66 [Back]
11 Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Reubinoff, Pera, Fong, Trounson and Bongso. Nature Biotechnology Vol 18 April 2000. [Back]
12 The Age (Melbourne) 4 June 2000 [Back]
13 Embryonic stem cell lines -- ibid [Back]
14 Neural progenitors from human embryonic stem cells" Reubinoff, BE; Itsykson, P, Turetsky, T; Pera, M. F; Reinhartz, E; Itzik, A; and Ben-Hur, T. Nature Biotechnology Vol 19 December 2001. [Back]
15 Testimony of Richard M. Doerflinger before the Subcommittee on Labor, Health and Human services, and Education, United States Senate Appropriation Committee: July 18, 2001 [Back]