Framing the Debates on Human Cloning and Human Embryonic Stem Cells: Pluripotent vs. TOTIPOTENT


Law Schools/Journals:

** Elizabeth C. Price, "Does the FDA Have Authority to Regulate Human Cloning?", Harvard Journal of Law and Technology, Vol. 11, No. 3, Summer 1998 (http://jolt.law.harvard.edu/articles/pdf/v11/11HarvJLTech619.pdf): [ftnt # 18, pg. 662] Somatic cell nuclear transfer should be distinguished from blastomere separation which involves the splitting of embryonic cells to form identical twins, triplets, or an even greater number of duplicates. NBAC REPORT. supra, at 15. Cloning by embryo splitting is common amongst animal breeders and was successfully performed on human embryos in 1993. See Gina Kolata, Scientist Clones Human Embryos, and Creates an Ethical Challenge, N.Y. TIMES, Oct. 24, 1993, at A 1; Rebecca Kolberg, Human Embryo Cloning Reported, 262 SCIENCE 652,652-53 (1993)."

** Ted Vosk, Human Cloning and FDA Regulation; Hutt, "Food and Drug Law", LEDA at Harvard Law School (#60472653) (http://leda.law.harvard.edu/leda/data/212/tvosk.html): "The third type of cloning can be broken down into two distinct processes; "blastomere separation" and "nuclear transplantation cloning". Blastomere separation involves the separation of embryonic cells, known as blastomeres, for use in producing multiple organisms which are genetically identical. Each blastomere is an undifferentiated cell and is totipotent. Totipotency indicates that each cell has the "total potential" to create an entire organism. The cells are separated soon after fertilization when the embryo consists of only two to eight cells and then implanted into the uterus of separate surrogate "parents" and allowed to develop normally in the womb. This form of cloning has great relevance for the livestock breeding industry and is not a cause of the current controversy."

** P. Campbell, G. Maranto, C. R. Cantor, L. H. Glantz, and F. H. Miller, "Gene Therapy: Legal, Financial and Ethical Issues", Boston University Journal of Science and Technology Law, March 20, 1997, pp. 18-19 (http://www.bu.edu/law/scitech/volume4/4jstl03.pdf): "Germ-line tinkering is the end to which these three lines of research that I mentioned earlier are headed. [Nuclear transplantation, genetic engineering, and reproductive medicine.] In 1983, when the first artificial twinning of horses was performed in this country using another type of cloning known as blastomere separation, [ftnt 110] ethicists insisted that no one would ever attempt the procedure on humans because there was too much opposition within ethical review boards and other institutional oversight bodies to permit it. They were wrong. In 1993, Jerry Hall at George Washington University Medical Center performed blastomere separation using 'genetically abnormal' human embryos. He told Science that he did it intentionally to 'get the ethical discussion moving.' The discussion did not 'move', however, just as it did not move in the late 1960s, when scientists issued the same assurance that cloning of anything was impossible and unthinkable. We should have known better, but too often in our society, we react only to what exists. Now, we have the unthinkable and we must scramble to catch up.

"[ftnt 110]: In relation to cloning, blastomere separation 'splits the cells or blastomeres of an early multicelled embryo before the cells have begun to differentiate. Because each blastomere at this stage is in theory totipotent (that is, capable of producing an entire organism itself), separated cells can become new embryos, all of which have the same genome.' John A. Robertson, "The Question of Human Cloning", Hastings Center Report, Mar. 1, 1994, at 6."

** George Annas, " II. Cloning and Imagination", in Human Cloning: A Choice or an Echo?, University of Dayton Law Review, winter 1998, Vol. 23, Num. 2, "Commentaries" (http://www.bumc.bu.edu/www/sph/lw/pvl/html3-variant.html): "Twinning by splitting an extracorporeal human embryo in two is the most rudimentary form of human cloning, and the closest to natural twins. The primary justification for embryo splitting has been to improve the efficiency of IVF, and the American Society for Reproductive Medicine ('ASRM') has justified research on embryo splitting as a possible way to improve the efficiency of IVF. ASRM's ethics committee cautions, however, that all twinned embryos should be implanted and gestated together to prevent the copy-original 'delayed twin' problem that is at the center of the cloning debate."

** [Re "nano-cloning human beings] Joel Rothstein Wolfson, "Social and Ethical Issues in Nanotechnology: Lessons from Biotechnology and Other High Technologies", 22 Biotechnology Law Report 376, No. 4 (August 2003), pp. 13-14 (http://www.blankrome.com/publications/Articles/WolfsonNanotechnology.pdf): "Nanotechnology can be used to clone machines as well as living creatures. Issues similar to those currently plaguing policy makers about biological cloning need to be raised early in the life of nanotechnology. ... Proponents of nanotechnology postulate a world where DNA strands can be custom built by repairing or replacing sequences in existing strands of DNA or even by building the entire strand, from scratch, one sequence at a time. With enough nanorobots working quickly enough, one could build a DNA strand that will produce a perfect clone. The same issues will arise, or re-arise, if nanotechnology is successful in promoting cloning of DNA segments, cells, organs, or entire organisms.

... It is likely that nanotechnology's efforts will lead to twists in the assumptions that lead to the resolution of cloning issues in terms of genetic bioengineering. Policy makers should anticipate, now, that in setting the boundaries for bioengineered cloning, the need to foresee issues that will arise from cloning by nanotechnology and be ready to reevaluate cloning regulation before nanotechnology perfects its own methods of cloning. If we do not anticipate the nanotechnology problems, the debate will emerge in an environment like the current one: one filled with a frenzy and uproar, rather than in an atmosphere of reflection and deliberateness."

IVF Center websites:

** Professor Dr. Mithhat Erenus, "Embryo Multiplication" (http://www.hekim.net/~erenus/20002001/asistedreproduction/micromanipulation/embryo_multiplication.htm): "In such cases, patients may benefit from embryo multiplication, as discussed in the study by Massey and co-workers. ... Since each early embryonic cell is totipotent (i.e., has the ability to develop and produce a normal adult), embryo multiplication is technically possible. Experiments in this area began as early as 1894, when the totipotency of echinoderm embryonic cells was reported ... In humans, removal of less than half of the cells from an embryo have been documented. No adverse effects were reported when an eighth to a quarter of the blastomeres were removed from an embryo on day 3 after insemination. ... Further evidence supporting the viability and growth of partial human embryos is provided by cryopreservation. After thawing four-cell embryos, some cells may not survive, leaving one-, two-, or three-cell embryos. These partial embryos survive and go to term, but at a lower rate than whole embryos. ... Based on the results observed in lower order mammals, the critical period of development to ensure success in separating human blastomeres should be at the time of embryonic gene expression, which is reported in humans to be between the four- and eight-cell stages. .... The second potential method of embryo multiplication is blastocyst splitting. ... Embryo multiplication by nuclear transfer has been used in experimental cattle breeding programs. ... IVF clinics routinely replace multiple (three to four) embryos into the uterus to increase the chances of a successful pregnancy. For couples who have less than three quality embryos for transfer, blastomere separation could be of benefit."

** The Twins Foundation (http://twinsfoundation.com/ru-v9n1-1994.htm): "New Ways to Produce Identical Twins -- A Continuing Controversy", 'Identical twins occur naturally approximately 3.5 times out of every 1000 human births. And, to date, scientists still don't know why and can't predict that they will, in any given birth, occur. However, in the last half of this century, and indeed, in the past ten to fifteen years, scientific advances have impacted on twins and other multiples and their families in numerous ways.

'Now, a new method of actually producing identical twins looms near. Called "blastomere separation" (the separation of a two- to eight-cell blastomere into two identical demi-embryos), it is potentially one method of helping infertile couples have children through in vitro fertilization (IVF).

'The following is excerpted from the medical journal Assisted Reproduction Reviews, May 1994. Dr. Joe B. Massey, who heads an in vitro clinic in Atlanta ... reviews the advances in blastomere separation and discusses the potential indications, benefits, limitations, and ethics of using this method to produce monozygotic twin embryos for IVF patients. The Twins Foundation, by presenting Dr. Massey's material for your information, neither advocates nor rejects any such procedures: "Embryo Multiplication by Blastomere Separation - One Doctor's Proposal" [Massey]. In spite of many advances in human vitro fertilization (IVF), there are still many problems. While leading clinics now have success rates of about 30%, many other clinics lag behind. Still, the number of couples undergoing IVF continues to increase despite high costs.

'According to Dr. Massey, "Observations on the potential impact of removing less than half of the cells from the human embryo have been well documented in pre-clinical embryo biopsy studies." (For more on this story see Research Update Vol. 9, No. 1, 1994).'

** Ethics Committee of the American Society for Reproductive Medicine (http://www.asrm.com/Media/Ethics/ethics94.html): "Ethical Considerations of Assisted Reproductive Technologies": Originally published as a supplement to the ASRM medical journal (Fertility and Sterility 1994;62:Suppl 1), Ethical Considerations for Assisted Reproductive Technologies covers the American Society for Reproductive Medicine's position on several aspects of reproductive medicine, including: ... the moral and legal status of the preembryo, ... the use of donor sperm, donor oocytes and donor preembryos, ... the cryopreservation of oocytes and preembryos, micro techniques such as: zona drilling, microinjection, blastomere separation (cloning), and assisted hatching ... "

** American Society of Reproductive Medicine (http://www.asrm.com/Media/Ethics/embsplit.html): "Because early embryonic cells are totipotent, the possibility of splitting or separating the blastomeres of early preimplantation embryos to increase the number of embryos that are available for IVF treatment of infertility is being discussed. Because embryo splitting could lead to two or more embryos with the same genome, the term "cloning" has been used to describe this practice. ... Splitting one embryo into two or more embryos could serve the needs of infertile couples in several ways. For couples who can produce only one or two embryos, splitting embryos could increase the number of embryos available for transfer in a single IVF cycle. Because the IVF pregnancy rate increases with the number of embryos transferred, it is thought that embryo splitting when only one or two embryos are produced may result in a pregnancy that would not otherwise have occurred. For couples who produce more than enough embryos for one cycle of transfer, splitting one or more embryos may provide sufficient embryos for subsequent transfers without having to go through another retrieval cycle, thus lessening the physical burdens and costs."

IVF textbooks:

** Peter Brinsden (ed.), A Textbook of In Vitro Fertilization and Assisted Reproduction, 2nd edition (New York: The Parthenon Publishing Group, 1999); Kay T. Elder, "Laboratory techniques: Oocyte collection and embryo culture", p. 197: "Surprisingly, fragmented embryos, repaired or not, do implant and often come to term. This demonstrates the highly robust nature of the human embryo, as it can apparently lose over half of its cellular mass and still recover."

** Geoffrey Sher, Virginia Davis, and Jean Stoess, In Vitro Fertilization: The A.R.T. of Making Babies (copyright 1998 by authors; information by contacting Facts On File, Inc., 11 Penn Plaza, New York, NY 10001): "(2) the fertilized egg, which has not yet divided, is now known as a zygote; (3) the egg begins to divide and is now known as an embryo; at this point each blastomere, or cell, within the embryo is capable of developing into an identical embryo." (p. 20)

On-line Encyclopedias:

** MSN Encarta Online Encyclopedia 2005, "Cloning: IV. How Scientists Clone Cells", Reviewed By: Ian Wilmut, Ph.D., D.Sc. Head, Department of Gene Expression and Development, Roslin Institute (http://encarta.msn.com/text_761567589___3/Cloning.html): "In the 1950s scientists began to experiment with embryo cells that were undifferentiated - that is, they had not yet specialized into a particular type of cell. Scientists found that such embryo cells are totipotent (able to give rise to all the different cell types in the body). Exploiting this characteristic, scientists developed three techniques to clone embryo cells: blastomere separation, blastocyst division, and somatic cell nuclear transfer.

"Blastomere Separation: In blastomere separation, scientists fertilize an egg cell with a sperm cell in a laboratory dish. The resulting embryo is allowed to divide until it forms a mass of about four cells. Scientists remove the outer coating of the embryo and place it in a special solution that causes the individual cells of the embryo, known as blastomeres, to separate. Scientists then put each blastomere in culture, where it forms an embryo containing the same genetic makeup as the original embryo. Each new embryo can then be implanted into the uterus of a surrogate mother to develop during a normal pregnancy.

"Blastocyst Division: In blastocyst division, scientists allow a fertilized egg to divide until it forms a mass of about 32 to 150 cells, known as a blastocyst. Scientists then split the blastocyst in two and implant the two halves into the uterus of a surrogate mother. The two halves develop as identical twins.

"Somatic Cell Nuclear Transfer: ... In this technique, scientists transfer the genetic material from a donor's somatic cell (any body cell other than an egg or sperm cell) to an enucleated egg cell—that is, an egg cell with its nucleus, and thus its genetic material, removed. The resulting cloned cell contains the genetic material of the donor's somatic cell. Scientists merge the somatic cell and enucleated egg cell using fusion or injection. In the fusion method, scientists place a somatic cell in contact with an enucleated egg cell. An electric pulse applied to the two cells pushes the somatic cell's nucleus into the enucleated egg cell. With the injection method, scientists inject the somatic cell's nucleus directly into the enucleated egg cell.

"In early experiments with somatic cell nuclear transfer, the procedure only worked using the nuclei from embryonic cells or cells from immature animals. In 1996 British scientists produced the sheep Dolly using a variation of somatic cell nuclear transfer that used the nuclei from adult cells. Scientists treated the adult donor cell to make it quiescent (less active) so that the genes of the adult cell behaved more like an undifferentiated embryo cell. They then isolated an udder cell from an adult sheep and starved the cell, forcing it into a resting stage that prevented the nucleus from dividing. Scientists found that this resting stage helps the adult cell return to an embryonic state. Scientists transferred the genetic material from the nucleus of the adult udder cell to an enucleated egg cell from a second sheep. The resulting embryo was then implanted into the uterus of a third sheep, where it developed during a normal pregnancy.

The birth of Dolly paved the way for cloning cells taken from adult animals, enabling scientists to choose the mature individual they want to duplicate. Using cells from immature animals makes it more difficult for scientists to predict with certainty the physical characteristics of the resultant clone.

"Somatic cell nuclear transfer only uses genetic material found in a donor cell's nucleus. But not all of an animal's genes are located in the nucleus. A few dozen genes reside in the mitochondria, a cell structure found outside of the nucleus in the cell's cytoplasm. As a result, clones derived from somatic cell nuclear transfer may have mitochondrial genes from the enucleated egg cell used in the cloning process, not just genes from the donor's genetic material."

On-line Magazines:

** Anita Jones (UVa., School of Engineering and Applied Science), "The Cloning Process", in "On Human Cloning: A Discussion", as published in Wired Magazine (March 1998), pg. 146 (http://www.cs.virginia.edu/~jones/tmp352/projects98/group9/tcp.html): "The world has entered a new technological era with the ability to clone. There are already multiple techniques of cloning that have been proposed for this new technology. The most popular of these are the Nuclear Transfer and the Blastomere Separation techniques. ...

"Blastomere Separation: For fear that the Nuclear Transfer technique continues to be inefficient, scientists have designed a new technique, Blastomere Separation, with the sole purpose of cloning human beings. The Blastomere Separation technique, or embryo splitting, first involves fertilizing an egg. The egg produces an embryo which, in the early stages of development, divides to form identical genetic embryos called blastomeres. The blastomeres' covering (the pellucida) is opened, and the blastomeres are removed. Each blastomere is protected by an artificial pellucida, and each blastomere develops into new embryo. Once the blastomere ceases separating, the embryo is stable and prepared to be implanted into the womb of the recipient mother. The number of identical embryos is unlimited because each extracted blastomere divides into multiple identical embryos, which can then be extracted to produce more clones. This both lowers the price and increases the efficiency of the cloning process.

"In this every-changing development of technology, anything is indeed possible, and with every insurmountable obstacle comes a new scientific miracle. Even if neither the Nuclear Transfer nor the Blastomere Separation techniques prove successful, eventually the scientific community will control the human cloning technology, and the world will have to be ready for it. (Non-online Source Material: Wired Magazine, March 1998, pg. 146)

Bioethics centers:

** Dr. Amin Abboud, "Cloning: All Life Matters" (September 2001), Media Commentaries, Australasian Bioethics Information (http://www.australasianbioethics.org/Media/RTL092001.htm): "There are two processes of animal cloning in a laboratory: blastomere separation and nuclear somatic transfer. Everyone is familiar with blastomere separation, because this is the process which results in identical twins. However, twinning is a chance occurrence in humans and other mammals. A single embryo separates into genetically identical halves at an early stage of development. The resulting offspring derive from a single zygote, which itself is the result of the fertilisation of one egg by one sperm. IVF reproduces this process in a laboratory. The developing embryo is split very soon after fertilisation when it is composed of two to eight cells. Each cell, called a blastomere, is totipotent, that is, it can grow into an individual organism. Totipotency allows scientists to split animal embryos into several cells which become genetically identical organisms. This capability has tremendous relevance to breeding cattle and other livestock. The process that resulted in Dolly, nuclear somatic transfer, is different. The nucleus of a cell from one individual is placed in the egg of another individual from which the nucleus has been removed. This nucleus can come from an embryo, foetus or adult (as in Dolly).

** Bioethics Curriculum Package - "Case 2: Cloning" [Northern California Biotechnology Center, (http://smccd.net/accounts/skyline/NCBC/bioethics/bioethics6.html): "Cloning refers to any attempt to produce a genetically identical copy of another organism. What this general definition leaves out, however, are the different means by which cloning is achieved. These are: ... In embryo splitting (also called twinning and blastomere separation), embryos cells are separated during the initial stages of cell division, before the cells have begun to differentiate. Since at this stage each cell is totipotent, capable of becoming any kind of tissue in the body, the cells have the potential of becoming new embryos, with genomes identical to the original embryo (Robertson, 1994, 6).

"In humans, embryo splitting is done between the 2 and 8-cell stage of division, on the second and third day of life. After this point the cells, at least those on the outside of the embryo, begin to differentiate, losing the potential to become any kind of tissue. These differentiated cells retain their entire genome, but only those parts needed for the cell's function are turned on.

"In nuclear transfer, the nucleus of a single cell (from an embryo, fetus, or adult) is removed and transferred into an unfertilized, enucleated egg. This can create a new, totipotent embryo, in which the donor nucleus supplies the genetic material and the egg's cytoplasm supplies the signals needed for an embryo to develop. (The donor egg also supplies a small amount of mitochondrial DNA in the cytoplasm, so the clone is not an exact genetic replication of the original donor.)

"For example, in the Roslin experiment, Dolly was created from a cell of breast tissue. Because this was an adult cell and had differentiated, only those parts of the genome important for breast cell functions were turned on. But once the nucleus of the breast tissue cell was transferred to an egg whose cytoplasm is designed to turn on the entire genome, the breast cell was able to reverse its differentiation and become totipotent. (Before transfer, the Roslin group had assisted this process by depriving the donor cell of needed nutrients. This put the cell into an inactive state, called quiescence, which meant that the cell was not replicating its DNA. This made it easier to transfer successfully.)

Nuclear transfer, then, demonstrates that parts of a cell's genome are not permanently inactivated in development, as previously thought. Rather the genome, even in an adult, retains the potential to create a genetically identical organism (Klotzko 128)."

Technology websites:

** "Cloning Technology: An Ongoing Battle between technologies versus ethical implication", Research and Markets (http://www.researchandmarkets.com/feats/download_pdf.asp?report_id=10805):
"3. Cloning Techniques
3.1. Cloning Process
3.1.1. Blastomere separation Process"

** "Cloning Fact Sheet", BioSino (http://www.biosino.org/pages/hot1.htm): "The possibility of human cloning, raised when Scottish scientists at Roslin Institute created the much-celebrated sheep 'Dolly' (Nature 385, 810-13, 1997), has aroused worldwide interest and concern because of its scientific and ethical implications. The feat, cited by Science magazine as the breakthrough of 1997, also has generated uncertainty over the meaning of 'cloning' -- an umbrella term traditionally used by scientists to describe different processes for duplicating biological material. ... "What is cloning? To Human Genome Project researchers, cloning refers to copying genes and other pieces of chromosomes to generate enough identical material for further study. Two other types of cloning produce complete, genetically identical animals. Blastomere separation (sometimes called "twinning" after the naturally occurring process that creates identical twins) involves splitting a developing embryo soon after fertilization of the egg by a sperm (sexual reproduction) to give rise to two or more embryos. The resulting organisms are identical twins (clones) containing DNA from both the mother and the father. Dolly, on the other hand, is the result of another type of cloning that produces an animal carrying the DNA of only one parent. Using somatic cell nuclear transfer, scientists transferred genetic material from the nucleus of an adult sheep's udder cell to an egg whose nucleus, and thus its genetic material, had been removed. (All cells that are not egg or sperm cells are somatic cells.)"

Various books:

** Daniel Svensson, Cloning [book], [Mimers Brunn - Online] (2003-04-12) (http://www.mimersbrunn.se/arbeten/2621.asp): "Scientists began to experiment with embryo cells that were undifferentiated, in the 1950s. Undifferentiated means that they were not yet specialized into a particular type of cell.

"Scientists found out that such embryo cells are totipotent (they are able to give rise to all the different cell types in the body). Scientists made use of this characteristic, and developed three different techniques to clone embryo cells: blastomere separation, blastocyst division, and somatic cell nuclear transfer.

"Blastomere Separation: In blastomere separation, scientists fertilize an egg cell with a sperm cell in a laboratory dish. The embryo is now allowed to divide itself, until it forms a mass of about four cells. After that, the outer layer of the embryo is removed and placed in a special solution that causes the individual cells of the embryo, which are known as blastomeres, to separate. Then each blastomere is set in culture, where it forms an embryo containing the same genetic makeup as the original embryo. Each new embryo can then be implanted into the uterus of a surrogate mother to develop during a normal pregnancy.

"Blastocyst Division [[blastocyst splitting]]: In blastocyst division, scientists allow a fertilized egg to divide until it forms a mass of about 32 to 150 cells, known as a blastocyst. After that the blastocyst is split in two parts and then both parts are implanted into the uterus of a surrogate mother. The two halves develop as identical twins.

"Somatic Cell Nuclear Transfer: While blastomere separation and blastocyst division produce animals containing the genetic material from both parents, somatic cell nuclear transfer produces an animal that carries the genetic material of only one parent."

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