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

University Scientific Research Centers:

** Genetic Science Learning Center, University of Utah (posted as of Feb. 6, 2005) ( "You might not believe it, but there are human clones among us right now. They weren't made in a lab, though: they're identical twins, created naturally. Below, we'll see how natural identical twins relate to modern cloning technologies. ... Cloning technologies have been around for much longer than Dolly, though. How does one go about making a genetic copy of an organism? There are a couple of ways to do this: artificial embryo twinning and somatic cell nuclear transfer. How do these processes differ?

"1. Artificial Embryo Twinning: Artificial embryo twinning is the relatively low-tech version of cloning. As the name suggests, this technology mimics the natural process of creating identical twins. In nature, twins occur just after fertilization of an egg cell by a sperm cell. In rare cases, when the resulting fertilized egg, called a zygote, tries to divide into a two-celled embryo, the two cells separate. Each cell continues dividing on its own, ultimately developing into a separate individual within the mother. Since the two cells came from the same zygote, the resulting individuals are genetically identical. ... Artificial embryo twinning uses the same approach, but it occurs in a Petri dish instead of in the mother's body. This is accomplished by manually separating a very early embryo into individual cells, and then allowing each cell to divide and develop on its own. The resulting embryos are placed into a surrogate mother, where they are carried to term and delivered. Again, since all the embryos came from the same zygote, they are genetically identical."

** Laboratoire de Didactique et Epistémologie des Sciences (LDES), Geneva University, Geneva, Switzerland, "Biology, Education and Ethics" ( "Since the 1st conference organized by the IUBS Commission for Biological Education (CBE) in 1975 in Upsala, Sweden, a lot of things have changed... During this last quarter century, the research in biology has led to an enormous expansion of our knowledge. For example, the ability to maintain cultures of totipotent human embryo cells points towards new directions in fundamental research."

U.S. Government:

** Linda Bren, "Cloning: Revolution or Evolution in Animal Production?", FDA Consumer Magazine (May-June 2003), FDA: US Food and Drug Administration ( "The Cloning Process: Early methods of cloning in the 1970s involved a technology called embryo splitting, or blastomere separation. Embryos were split into several cells and then implanted into a surrogate mother for growth and development. But there were a limited number of splits that could be made, and only a few clones could be produced from one egg. The characteristics of the clone were also unpredictable because scientists were cloning from an embryo whose traits could not be predicted."

** National Institutes of Health, Office of Science Planning and Policy, "CLONING: Present Uses and Promises" (April 27, 1998) ( "Cloning and somatic cell nuclear transfer are not synonymous. ... Cloning can be successfully accomplished by using a number of different technologies. Somatic cell nuclear transfer is one specific technology that can be used for cloning. It is important to note that the use of somatic cell nuclear transfer is not limited to cloning an organism; there are other potential applications for both research and medicine that are discussed in this paper."

** National Institutes of Health Report on Stem Cells (June 17, 2001) ( "After fertilization, the zygote makes its way to the uterus, a journey that takes three to four days in mice and five to seven days in humans. As it travels, the zygote divides. The first cleavage produces two identical cells and then divides again to produce four cells. If these cells separate, genetically identical embryos result, the basis of identical twinning." (p. A-3)

** Library of Congress: Judith A. Johnson, "Human Cloning", National Council for Science and the Environment, Congressional Research Service Report #RL313558, February 25, 2002, pp. 7, 11 ( "The NIH Panel also identified areas of human embryo research it considered to be unacceptable, or to warrant additional review. It determined that certain types of cloning [11] without transfer to the uterus warranted additional review before the Panel could recommend whether the research should be federally funded.

"[Footnote 11]: These were blastomere separation, where a two-to eight-cell embryo is treated causing the cells (blastomeres) to separate; and, blastocyst division, in which an embryo at the more advanced blastocyst stage is split into two."

** Library of Congress: Irene Stith-Coleman, "Human Embryo Research", National Council for Science and the Environment, Congressional Research Service [Library of Congress] Report #95-910, December 1, 1998 ( "Areas of research considered by the Panel to warrant additional review include studies which would use fetal oocytes for fertilization and investigations involving the development of embryonic stem cells (which could potentially improve bone marrow transplantation) from embryos fertilized specifically for this purpose. In addition, cloning by blastomere or blastocyst splitting without transfer to a patient's uterus were considered in need of further review. The Panel did not determine if such proposals were acceptable or not acceptable, but suggested that they not be federally funded for the foreseeable future, unless there was an extraordinary demonstration of scientific or therapeutic justification, as well as review by an ad hoc advisory body.

"(Footnote #17): In blastomere splitting, at the two- to eight-cell embryo stage, each cell is separated and cultured individually where they divide and form smaller-than-normal embryos, which can then be transferred to the uterus. In blastomere separation, a single embryo at the blastocyst stage is mechanically split into two and both halves are transferred to the uterus, where, identical twins could develop."

** Library of Congress: Irene Stith-Coleman, "Cloning: Where Do We Go From Here?", National Council for Science and the Environment, Congressional Research Service [Library of Congress] Report #97-335, April 23, 1998 ( "In Blastomere Separation, the outer coating, or zona pellucida, is removed from around a 2- to 8-cell embryo, then placed in a special solution that causes the cells, called blastomeres, to separate. Each cell can then be cultured individually, because at this stage of embryo development, each cell is totipotent, that is, it is undifferentiated and can develop into an organism. After dividing a few times, each blastomere may develop into a smaller-than-normal embryo that can be transferred to the uterus.

"In Blastocyst Division, also called induced twinning, an embryo, at the blastocyst stage, a more advanced stage of development than the blastomere, is mechanically split into two. The two parts can be transferred to the uterus. If both halves develop, then, at most, one blastocyst gives rise to identical twins.

"In Nuclear Transplantation, a nucleus is transferred from each blastomere of a 4- to 8-cell or later-stage embryo into the cytoplasm (cell contents other than nucleus) of an egg from which the genetic material has been removed (enucleated egg). To do this, the blastomere is placed beside an enucleated egg and their membranes are fused together artificially for example, with electrical pulses. The nucleus from the blastomere enters the egg cytoplasm and directs development of the embryo.

"The Scottish scientists used a variant of the nuclear transplantation technique, where the nucleus that programmed the creation of Dolly was transferred from the adult sheep mammary cell, not an embryo. Researchers had thought that when cells became differentiated to do certain jobs in the body, they could not revert to the embryo stage. For example, they thought that a cell that became a liver cell remained a liver, but that belief was disproved by the Scottish scientists. They were able to reprogram the genes in a mammary-gland cell to make it act like an undifferentiated embryo, which then developed into a sheep.

"(footnote #5: The information describing the 3 cloning techniques was obtained from The NIH Report of the Human Embryo Research Panel, September 1994.)"

State Commissions:

[[Note: The following California Report was strongly advocated by physician researcher Irving Weissman et al, and helped to successfully pass two laws in California prior to the passing of Proposition 71 - a law on human cloning, and a law on human embryonic stem cell research. The Report contains the same erroneous definitions of "cloning" and "stem cell research" advocated by Weissman et al, which are incorporated into two commission reports of the National Academy of Sciences which Weissman chaired as well.]]

** Report of the California Advisory Committee on Human Cloning: Cloning Californians?, Markkula Center for Applied Ethics, Santa Clara University, Sacramento, CA; January 11, 2002 ( "The Committee was given a relatively narrow mandate - to make recommendations about state policy on human cloning. Even that mandate, however, led to some difficult new issues. The most troublesome for the Committee was "cloning" by embryo-splitting (also known as "blastomere-separation"). Embryos can split at an early stage to produce two (or, rarely, more) people who are genetically identical. This is the source of human identical twins, who make up about one birth in every 240. ... there seems to be no substantial barrier to splitting embryos that are created through in vitro fertilization. In essence, this process would create additional identical twins. ... Embryo splitting is not addressed by the existing California legislation on cloning, nor did the Committee believe that it was clearly within our mandate. Embryo splitting, however, can raise issues similar to those arising from human reproductive cloning by nuclear transfer and addressed above. ...

"A second cluster of issues arises from the potential use of embryo-splitting to have a 'delayed' twin. One or more embryos created by this splitting could be frozen for a period of time, later to be removed from cryopreservation and implanted into a woman. This might occur weeks or years after the first embryo is implanted and brought to term. The latter twin would have a genotype that has already come into existence. The parents (or others) could wait and see whether they wanted another copy of the first child, producing many of the concerns of nuclear transfer cloning. ... The Committee was not charged with making recommendations on embryo-splitting, and thus did not solicit witnesses on the topic."

National Commissions (Reports and testimonies):

** Cloning Human Beings: Report and Recommendations of the National Bioethics Advisory Commission, Rockville, MD, June 1997 ( "Chap. 1, pg. 1, Intro: ... This technique of transferring a nucleus from a somatic cell into an egg is an extension of research that had been ongoing for over 40 years using nuclei derived from non-human embryonic and fetal cells. The demonstration that nuclei from cells derived from an adult animal could be "reprogrammed," or that the full genetic complement of such a cell could be reactivated well into the chronological life of the cell, is what sets the results of this experiment apart from prior work.

"Chap. 2 (pg. 13) ... The term "cloning" is used by scientists to describe many different processes that involve making duplicates of biological material. ... Although a single adult vertebrate cannot generate another whole organism, cloning of vertebrates does occur in nature, in a limited way, through multiple births, primarily with the formation of identical twins. However, twins occur by chance in humans and other mammals with the separation of a single embryo into halves at an early stage of development. ... Cloning of animals can typically be divided into two distinct processes, blastomere separation and nuclear transplantation cloning. ...

"(pg. 14) In blastomere separation, the developing embryo is split very soon after fertilization when it is composed of two to eight cells (see figure 1). Each cell, called a blastomere, is able to produce a new individual organism. These blastomeres are considered to be totipotent, that is they possess the total potential to make an entire new organism. This totipotency allows scientists to split animal embryos into several cells to produce multiple organisms ... This capability has tremendous relevance to breeding cattle and other livestock. ...

"(pg. 19) To reprogram the gene expression of a somatic cell it is not essential to fuse it with an egg; in some cases re-programming can occur through fusion of two adult cells. Cell fusion experiments, in which different somatic cell types are fused, have demonstrated that extensive reprogramming of differentiated nuclei can occur. ... These studies have further demonstrated that the stability of the differentiated state is not absolute. Thus, given the appropriate regulatory molecules and enough time to reprogram an adult nucleus, somatic cells can re-initiate earlier programs of differentiation. ...

"(pg. 31) Assisted Reproduction: ... In those instances where both individuals of a couple are infertile or the prospective father has non-functional sperm, one might envision using cloning of one member of the couple's nuclei to produce a child.

"APPENDIX A: GLOSSARY: Blastomere separation: a technique by which a jelly-like substance is removed from around a two-to eight-cell embryo, or morula, and the embryo is incubated in a special solution so that the blastomeres separate and fall apart. The blastomeres are then cultured separately."

** Daniel J. Kevles (Yale University) and Karen Hauda (U.S. Patent and Trademark Office), "Patentability of Human Organisms 1: History and Current Law", President's Bioethics Council, Fourth meeting (June 20, 2002) Session 5: Regulation 3 ( "Cloning can occur naturally, such as in identical twins or triplets. Additionally, there are two quite different artificial laboratory procedures aimed at duplicating embryos that have been termed cloning. ... Blastomere separation and nuclear transplantation: Also known as embryo splitting, blastomere separation is an artificial cloning procedure that involves isolating individual cells of very early embryos, and growing them into separate embryos. Blastomere separation has been used successfully to increase the pregnancy rate in cattle. Nuclear transplantation is an artificial cloning technique that involves removing the nucleus from a cell and inserting the nucleus into an unfertilized egg from which the nucleus has been removed, an enucleated cell."

International Commissions/Laws:

** European Parliament: Professor Andrew Webster, Science and Technology Studies Unit (University of York), Dr. Nik Brown, SATSU (University of York), Dr. Brian Rappert, SATSU (University of York), Dr. Paul Martin, Institute for the Study of Genetics Biorisk and Society (University of Nottingham), Rob Frost (University of Nottingham.), Dr. Adam Hedgecoe, Department of Science and Technology Studies (UCL), Human Genetics: An Inventory of New and Potential Developments in Human Genetics and Their Possible Uses - Final Study, Scientific and Technological Options Assessment (STOA) [European Parliament, Directorate General for Research, Directorate, Luxembourg, September 2001 - PE 303.129/Fin St], pp. 45, 46 ( "Notwithstanding these notes of caution, we can refer to a number of processes as 'cloning':

"1.1 Embryo Splitting (Blastomere Separation): Embryo splitting is a method whereby the cells of a very early embryo (2, 4 or 8 cell stage) are separated out to continue developing individually. Since at this stage each blastomere is 'totipotent', the undifferentiated cell will continue to divide, then differentiate into functionally specific cell types and eventually develop into a complete organism. Each of the resulting organisms will be genetically identical. This process of embryo splitting is, in many respects, a technically induced means by which identical twins (AKA monozygotic twins) occur naturally.

"In the context of human reproductive fertility, this form of cloning was first used experimentally in 1992 when researchers split the cells of 17 chromosomally abnormal human embryos to determine if development would continue. Whilst there was no intention to re-implant the developing embryos to develop in vivo, it nevertheless demonstrated the application of the technique in human reproduction. Numerous infertility clinics are capable of producing clones in this way. ...

"1.2 Nuclear Transfer: Until the events surrounding Dolly in 1997, nuclear transfer in mammals had only been accomplished by inserting an undifferentiated embryonic cell into either a fertilised zygote or egg from which the nucleus had been removed. This has been possible in sheep since the mid 1980s. In the year preceding the announcement of Dolly, the Roslin Institute published findings from a nuclear transfer procedure resulting in the birth of two genetically identical sheep, Megan and Morag. The key difference between the 1996 and 1996 events is the source of nuclear donation. In the 1996 case, the nuclear source was an embryonic sheep cell. Dolly on the other hand was derived from an already adult somatic and differentiated cell. ...

"1.3 Somatic Cell Nuclear Transfer (SCNT): The announcement by the Roslin Institute in 1997 represents the first instance of using a somatic nuclear source to produce a cloned adult mammal. Dolly was derived from cells taken from the udder of a 6-year-old ewe. 277 such cells were then cultured in vitro and fused with unfertilised eggs from which the nucleus had been removed. This process resulted in 29 viable reconstructed eggs, each with a nucleus from the adult animal, which was then implanted into surrogate ewes. Only one of these would result in a viable adult mammal, Dolly.

"The technical shift from embryonic to somatic nuclear transfer holds the possibility of much greater flexibility in the production of cloned and transgenic mammals. By using treated and cultured somatic cells, researchers need not be overly restricted by having to rely on a limited number of available embryonic cells."

** Council of Europe: Anne Mc Laren (Wellcome/CRC Institute - UK), "Part Five: Research on Embryos In Vitro", in Medically Assisted Procreation and the Protection of the Human Embryo and Foetuses, 3rd Symposium on Bioethics, Council of Europe (Legal Affaires) ( "The various types of research: implications and objectives of research, present and future research on embryos in vitro: ...

"Warranting additional review:

** The Council of Europe: Human Cloning Regulation in Europe [in American Center for Law and Justice, Info Letters, CFJD MEMO, 2001-03-09] ( "The member States of the Council of Europe, the other States and the European Community Signatories to this Additional Protocol to the Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine, ... Noting scientific developments in the field of mammal cloning, particularly through embryo splitting and nuclear transfer ..."

** Australia: The Cloning of Humans (Prevention) Bill 2001 (Queensland) "Cloning can occur naturally in the asexual reproduction of plants, the formation of identical twins and the multiplication of cells in the natural process of repair. The cloning of DNA, cells, tissues, organs and whole individuals is also achievable with artificial technologies. ... The cloning of a cell or an individual may be achieved through a number of techniques, including: molecular cloning ..., blastomere separation (sometimes called "twinning" after the naturally occurring process that creates identical twins): splitting a developing embryo soon after fertilisation 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. ... somatic cell nuclear transfer: the transfer of the nucleus of a somatic cell into an unfertilised egg whose nucleus, and thus its genetic material, has been removed. A number of scientific review bodies have noted that the term "cloning" is applicable in various contexts, as a result of the development of a range of cloning techniques with varying applications".

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