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

IX: Selected Bigliography on the Totipotency of Early Human Embryonic Stem Cells:

Human Embryology Textbooks:

[Note: there are many human embryology textbooks now available on-line, including several of those quoted directly here. For URLs, see endnotes below.9 Emphases have been used to aid those unfamiliar with the science.]

** Ronan O'Rahilly and Fabiola Muller, Human Embryology & Teratology (3rd ed.) (New York: Wiley-Liss, 2001): "Biopsy of an embryo can be performed by removing one cell from a 4-cell, or two cells from an 8-cell, embryo. This does not seem to decrease the developmental capacity of the remaining cells. ... The embryo enters the uterine cavity after about half a week, when probably at least 8-12 cells are present and the endometrium is early in its secretory phase. Each cell (blastomere) is considered to be still totipotent (capable, on isolation, of forming a complete embryo), and separation of these early cells is believed to account for one-third of cases of monozygotic twinning." (p. 37)

** O'Rahilly and Muller (2001), p. 136-137: "Stem cells comprise a small subpopulation of multipotent or pluripotent, ultrastructurally unspecialized, slow-cycling cells that possess the ability of self-renewal and can produce cells that are destined to differentiate. (In contrast, primordial germ cells and those of a morula are totipotent: i.e., they can develop into any type of embryonic tissues and can even form an entirely new embryo). Stem cells have a long proliferative reserve and are greatly influenced by their microenvironment. The signals that lead a stem cell to follow a specific developmental pathway are being investigated. When they become unipotent, i.e., committed to one line of development, the cells are generally known as progenitors and then precursors of differentiated cells. Stem cells are characteristic of all self-renewing tissues, such as the blood and the epidermis. Those in adult bone marrow include hematopoietic cells and mesenchymal cells; the latter can differentiate into fat, cartilage, bone, muscle, and possibly other types (e.g., hempatic cells). Dormant neural stem cells are present in the adult mammalian brain (e.g., in the ependyma, Rao, 10999) and are capable of forming neurons and glia. In addition to embryonic stem cells from preimplantation blastocysts, stem cells can be obtained from an adult. These latter possess remarkable potentialities; e.g., brain cells can become blood cells, and cells from bone marrow can become hepatic cells. Ethical concerns are intensified by the experimental finding in primates (in contrast to the mouse) that embryonic stem cells are totipotent and can develop into a complete embryo with a primitive streak."

** Bruce Carlson, Human Embryology & Developmental Biology (St. Louis, MO: Mosby, 1999) (2nd ed.): "Mammalian embryogenesis is considered to be a highly regulative process. Regulation is the ability of an embryo or an organ primordium to produce a normal structure if parts have been removed or added. [[i.e., regulation can "heal" a damaged embryo.]] At the cellular level, it means that the fates of cells in a regulative system are not irretrievably fixed and that the cells can still respond to environmental cues. Because the assignment of blastomeres into different cell lineages is one of the principal features of mammalian development, identifying the environmental factors that are involved is important. (pp. 44-49); ... Of the experimental techniques used to demonstrate regulative properties of early embryos, the simplest is to separate the blastomeres of early cleavage-stage embryos and determine whether each one can give rise to an entire embryo. This method has been used to demonstrate that single blastomeres, from two- and sometimes four-cell embryos can form normal embryos, [[i.e., regulation can also revert totipotent embryonic cells back to new human organisms.]] ... (p. 44); ... Another means of demonstrating the regulative properties of early mammalian embryos is to dissociate mouse embryos into separate blastomeres and then to combine the blastomeres of two or three embryos. The combined blastomeres soon aggregate and reorganize to become a single large embryo, which then goes on to become a normal-appearing tetraparental or hexaparental mouse. By various techniques of making chimeric embryos, it is even possible to combine blastomeres to produce interspecies chimeras (e.g., a sheep-goat). (p. 45); ... Blastomere removal and addition experiments have convincingly demonstrated the regulative nature (i.e., the strong tendency for the system to be restored to wholeness) of early mammalian embryos. Such knowledge is important in understanding the reason exposure of early human embryos to unfavorable environmental influences typically results in either death or a normal embryo. (p. 46); ... Some types of twinning represent a natural experiment that demonstrates the highly regulative nature of early human embryos, ... (p. 48); ... Monozygotic twins and some triplets, on the other hand, are the product of one fertilized egg. They arise by the subdivision and splitting of a single embryo. Although monozygotic twins could ... arise by the splitting of a two-cell embryo, it is commonly accepted that most arise by the subdivision of the inner cell mass in a blastocyst. Because the majority of monozygotic twins are perfectly normal, the early human embryo can obviously be subdivided and each component regulated to form a normal embryo." (p. 49)

** William J. Larsen, Essentials of Human Embryology (New York: Churchill Livingstone, 1998): [Monozygotic twinning in humans] "If the splitting occurred during cleavage -- for example, if the two blastomeres produced by the first cleavage division become separated -- the monozygotic twin blastomeres will implant separately, like dizygotic twin blastomeres, and will not share fetal membranes. Alternatively, if the twins are formed by splitting of the inner cell mass within the blastocyst, they will occupy the same chorion but will be enclosed by separate amnions and will use separate placentae, each placenta developing around the connecting stalk of its respective embryo. Finally, if the twins are formed by splitting of a bilaminar germ disc, they will occupy the same amnion." (p. 325)

** Moore and Persaud, The Developing Human: Clinically Oriented Embryology [Philadelphia: Saunders, 2003), 7th ed.]: [T]wins that originate from one zygote are monozygotic(MZ) twins or identical twins. The fetal membranes and placentas vary according to the origin of the twins. In the case of MS twins, the type of placenta and membranes formed depends on when the twinning process occurs. (p. 144). ... MZ twinning usually begins in the blastocysts stage, around the end of the first week, and results from division of the embryoblast into two embryonic primordia. Subsequently, two embryos, each in its own aminiotic sac, develop within the same chorionic sac and share a common placenta. ... [E]arly separation of embryonic blastomeres (e.g., during the two- to eight-cell stages) results in MX twins with two aminions, two chorions, and two placentas that may or may not be fused. (p. 147) ... About 35% of MZ twins result from early separation of the embryonic blastomeres; i.e., during the first 3 days of development. The other 65 % of MZ twins originate at the end of the first week of development. Late division of early embryonic cells, such as division of the embryonic disc during the second week, results in MZ twins that are in one amnionic sac and one chorionic sac. ... If the embryonic disc does not divide completely, or adjacent embryonic discs fuse, various types of conjoined MZ twins may form. (p. 148) ... Monozygotic twins .. represent about one-third of all twins; they are derived from one zygote. (pp. 151, 153)

Genetics textbooks:

** Tom Strachan and Andrew P. Read, Human Molecular Genetics 2 (New York: John Wiley & Sons, Inc, 1999): "Animal clones occur naturally as a result of sexual reproduction ... For example, genetically identical twins are clones who happened to have received exactly the same set of genetic instructions from two donor individuals, a mother and a father. A form of animal cloning can also occur as a result of artificial manipulation to bring about a type of asexual reproduction. The genetic manipulation in this case uses nuclear transfer technology: a nucleus is removed from a donor cell then transplanted into an oocyte whose own nucleus has previously been removed. The resulting 'renucleated' oocyte can give rise to an individual who will carry the nuclear genome of only one donor individual, unlike genetically identical twins. The individual providing the donor nucleus and the individual that develops from the 'renucleated' oocyte are usually described as "clones", but it should be noted that they share only the same nuclear DNA; they do not share the same mitochondrial DNA. ... Wilmut et al (1997) reported successful cloning of an adult sheep. For the first time, an adult nucleus had been reprogrammed to become totipotent once more, just like the genetic material in the fertilized oocyte from which the donor cell had ultimately developed. ... Successful cloning of adult animals has forced us to accept that genome modifications once considered irreversible can be reversed and that the genomes of adult cells can be reprogrammed by factors in the oocyte to make them totipotent once again." (pp. 508-509)

** Benjamin Lewin, Genes VII [Oxford: Oxford University Press and Cell Press, 2000), p. 605] [re why germ line cells are initially totipotent]: A change in methylation pattern occurs during embryogenesis. All allelic differences are lost when priordial germ cells develop in the embryo; irrespective of sex, the previous patterns of methylation are erased, and a typical gene is then unmethylated. The methylation pattern of germ cells is therefore established by a two stage process: first the previous pattern is erased by a genome-wide demethylation; then the pattern specific for each sex is imposed.

Scientific journals:

** DeMayo FJ, Rawlins RG, Dukelow WR, "Xenogenous and in vitro fertilization of frozen/thawed primate oocytes and blastomere separation of embryos", Fertil Steril. 1985 Feb;43(2):295-300, (PMID: 3967788) ( "Little research has been done on the in vitro and xenogenous fertilization of cryopreserved primate oocytes. This study reports the development of freezing and thawing methods for squirrel monkey oocytes with subsequent successful fertilization by these two methods. Preliminary results on techniques for blastomere separation using the hamster and squirrel monkey as models are also given. These studies have important implications relative to the long-term frozen storage of human oocytes, their subsequent thawing, in vitro fertilization and embryo transfer, and the use of the blastomere separation procedure, in conjunction with in vitro fertilization, in the diagnosis of embryonic normality and possible congenital defects prior to implantation."

** [Cloning researcher] Geraedts JP, de Wert GM., "Cloning: applications in humans; Technical aspects", Ned Tijdschr Tandheelkd. 2001 Apr;108(4):145-50]; [PMID: 11383357] ( "Cloning is possible by nucleus transplantation and by embryo splitting. Nucleus transplantation does not result in a genetically completely identical individual because the mitochondrial DNA originates from the ovum donor. Embryo splitting may be regarded as the artificial production of a monozygotic multiplet,"

** DeRenzo C, Seydoux G, "A clean start: degradation of maternal proteins at the oocyte-to-embryo transition", Johns Hopkins School of Medicine, Trends Cell Biol. 2004 Aug;14(8):420-6 [PMID: 15308208] ( "In this article, we explore the hypothesis that the coordinated degradation of germline proteins is essential for remodeling the oocyte into a totipotent zygote that is capable of somatic development."

** Don P. Wolf, "Assisted reproductive technologies in rhesus macaques", Reproductive Biology and Endocrinology, 2004, 2:37 [doi:10.1186/1477-7827-2-37] ( "ICSI produced embryos were used in efforts to create monozygotic twins by blastomere separation or blastocyst splitting. While pregnancies were achieved following the transfer of demi-embryos, only one was a twin and it was lost to spontaneous abortion. ICSI produced embryos have also served as the source of blastocysts for the derivation of embryonic stem cells. ... This effort has also achieved the first twin pregnancies in rhesus monkeys, the first non-human primate infants produced by nuclear transfer of embryonic cells, the first rhesus monkey infant born following the transfer of an ICSI-produced blastocyst employing a non-surgical procedure, the first monkey live birth resulting from the transfer of a demi-embryo created by blastomere separation at the 2-cell stage or blastocyst bisection and the first infants produced following laparoscopic embryo transfer. Recently we reported the outcomes of 87 pregnancies."

** "Cloning: From DNA Molecules to Dolly", Human Genome News (January 1998; 9:(1-2)) ( "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.

** Bukovsky A, Svetlikova M, Caudle MR., "Oogenesis in cultures derived from adult human ovaries", Reprod Biol Endocrinol. 2005 May 5;3(1):17 ( "Development of numerous mature oocytes from adult ovarian stem cells in vitro offers new strategies for the egg preservation, IVF utilization, and treatment of female infertility. In addition, other clinical applications aiming to utilize stem cells, and basic stem cell research as well, may employ totipotent embryonic stem cells developing from fertilized oocytes."

** Christopher R. Cogle, MD; Steven M. Guthrie, BS; Ronald C. Sanders, MD; William L. Allen, JD, Edward W. Scott, Ph.D.; Bryon E. Petersen, Ph.D., "Stem Cell Research: An Overview of Stem Cell Research and Regulatory Issues, Mayo Clin Proc. 2003;78:993-1003 ( "As an extension of research with embryonic stem cells, investigators have also used these 'pluripotent' cells to produce identical offspring. Three approaches have been used to clone progeny. The first technique is blastomere separation. Splitting blastomeres at this totipotent stage leads to the development of genetically identical offspring but can produce only a limited brood due to the low cell number at the blastocyst stage."

** Sills ES, Tucker MJ, Palermo GD. (Georgia Reproductive Specialists LLC, Atlanta 30342, USA), "Assisted reproductive technologies and monozygous twins: implications for future study and clinical practice", 1: Twin Res. 2000 Dec;3(4):217-23 (PMID: 11463142, at: "In vitro fertilization (IVF) relies on necessary (and, in some cases extended) embryo culture techniques potentially creating subtle ZP changes and subsequent MZ [monozygotic] twinning. With growing experience in the assisted reproductive technologies and particularly IVF, some preliminary reports have noted an increased frequency of MZ twins after procedures that artificially breach the ZP (i.e., intracytoplasmic sperm injection [ICSI], or 'assisted hatching').

** "Human Genome: The Book of Life", Science, Volume 3 No.5 (September-November 2000) ( "Cloning: For 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 creates identical twins (clones) which involves splitting a developing embryo soon after fertilization of the egg by a sperm to give rise to two or more embryos."

** George J. Annas, Arthur Caplan & Sherman Elias, "Stem cell politics, ethics and medical progress", Nature Medicine 5, 1339 - 1341 (1999) [doi:10.1038/70900] ( "Tremendous controversy has surrounded efforts to undertake research on totipotent human stem cells. To date public policy in the United States has attempted to skirt the ethical and social questions raised by this research. Annas et al. argue that research using human embryos as a source of totipotent stem cells can secure broad public support if there is an open and public discussion about the ethical justification for undertaking such research and the assurance of adequate federal regulation and oversight."

** "Rhesus monkey is model for human embryonic stem cell research", Reproduction and Development (Sept. 17, 2004), Wisconsin National Primate Research Center, Univ. of Wisconsin-Madison ( "The utility of MHC-defined and genetically identical animals in a viral challenge model would have tremendous impact on our ability to assess vaccine efficacy. This past year we have produced the first MHC-defined rhesus monkeys using assisted reproductive technologies. In addition, we have initiated studies to develop techniques for producing genetically identical MHC-defined monkeys using blastomere separation, or embryo splitting. Using these techniques, we have produced genetically identical blastocysts in vitro at a relatively high rate. Continued improvements in assisted reproductive technologies in rhesus monkeys will enable us to develop a unique animal production program for the creation of MHC-defined and genetically identical monkeys for use in immunological research and vaccine trials."

Scientific/Medical Professional Societies:

** American Medical Association: Cloning and Embryo Research: Report 7 of the Council on Scientific Affairs (A-99), (June 1999) ( "NOTE: This report represents the medical/scientific literature on this subject as of June 1999. ... Resolution 528, introduced at the 1997 Annual Meeting by the Section on Medical Schools and referred to the Board of Trustees by the House of Delegates, asks that: "The American Medical Association (AMA) work with the National Bioethics Advisory Commission and members of Congress and the executive branch to ensure that any legislation regulating human somatic cell nuclear transfer cloning not interfere with other significant important ongoing medical research;' and further, 'study the scientific and bioethical implications of research involving the earliest stages of human embryonic development, including somatic cell nuclear transfer cloning.'

"This report summarizes the scientific basis of cloning and describes its potential risks and benefits for clinical medicine and biomedical research. In this report, unless otherwise indicated, "cloning" will refer specifically to the production of genetically identical individuals via somatic cell nucleus transfer. This technique is one of four potential ways of cloning mammals ...

"Cloning Methods: All multicellular organisms are composed of two major categories of cells: (1) germ cells or gametes, which function in sexual reproduction; and (2) somatic cells, i.e., any cell that is not involved in sexual reproduction. Sexual reproduction usually requires the fusion of gametes from different genders. Some simple invertebrates and most plants reproduce asexually. Asexual reproduction involves either parthenogenesis (regrowing an entire organism from a piece of the donor) or cloning (the generation of an entire organism from a somatic cell). Vertebrates do not have the natural ability to reproduce asexually. ...

"Dolly was cloned via a technique referred to as 'somatic cell nuclear transfer' or 'somatic cell nuclear transplantation.' Before describing the technique in more detail, the other possible cloning methods are described. Post-fertilization development in mammals occurs as follows: ... "Early blastomeres are totipotent (from totipotency: the ability to form any cell type in the body), and therefore capable of regenerating an entire organism on their own in utero. It is thus possible to clone mammals by teasing apart the early blastomeres of an early embryo, reimplanting them in the uterus, and allowing each to develop independently. Scientists believe blastomere separation is one of the processes by which monozygotic twins or multiples normally form. It is also possible to divide the blastocyst in two (blastomere halving), to reimplant the two portions in the uterus, and to allow each half to develop independently into a complete organism. This is believed to be another way twins normally form ...

"All the genes of a cell (except the mitochondrial genome) are located in the nucleus. ... Since an oocyte is certainly totipotent, the best test for its cytoplasm's ability to confer totipotency onto a foreign nucleus is to transplant the foreign nucleus into it. ... However, complete development of a so-called "reconstructed embryo" to term, or better, adulthood, after a nuclear transfer experiment of this kind would constitute proof that both (1) the transplanted nucleus retains the potential for totipotency; and (2) the oocyte's cytoplasm can provide all the signals necessary to express the totipotency of the transplanted nucleus. This is precisely the result obtained by Wilmut and colleagues.

"Experimental evidence from studies on animal cell development suggests that the less differentiated a cell is (or the younger a cell is), the more extensive its developmental potential; sometimes as extensive as totipotency for some embryonic cells. ... It is, therefore, not surprising that the first successfully cloned mammalian farm animal (a sheep) [not Dolly] was derived from a cell of the inner cell mass of an embryo. Its birth constituted proof that at least some post-cleavage stage embryonic sheep cells are totipotent. The experiment was later successfully repeated using bovine embryonic cells, showing that the same was true in at least one other mammalian species. This success was followed by others, using cells previously considered unlikely to be returned to a totipotent state: (1) cloned lambs were derived from embryonic cells that had been cultured for a short time before being used as nuclei donors; (2) cloned lambs were derived from a line of cells that had been cultured for a longer time; and finally (3) cloned lambs, e.g., Dolly, were derived from fetal and from adult mammalian cells ...

"The apparent totipotency of G0-arrested nuclei also suggests that G0 may be a state that favorably predisposes a nucleus to be "reprogrammed" by the host cytoplasm, whatever its degree of differentiation immediately preceding the induction of quiescence. ...

"The nucleus is commonly viewed as the most important element in heredity; however, there are inherited human diseases caused by defects in the DNA of mitochondria. An example of an inheritable mitochondrial disease is MELAS (Mitochondrial myopathy Encephalopathy, Lactic Acidosis, Stroke-like episode). ...

One form of natural cloning already exists in human beings: the formation of identical (i.e., homozygotic) twins. Homozygotic twins (or multiples such as triplets, quadruplets, etc.) are produced when the first (or immediately subsequent) division of a fertilized egg develops into two (or more) cells that separate and each give rise to a complete individual. Alternatively, twins can arise from division of the blastocyst. Due to its similarity to twin formation, cloning has been called "delayed twinning," because it is a means of producing a twin for an organism at a later point than twinning would naturally occur."

** Society for Developmental Biology: Leland G. Johnson, Johnson & Volpe's Patterns & Experiments in Developmental Biology (3rd ed.), (McGraw-Hill Co., Inc., 2001) ( "The simpler and more effective procedure for blastomere separation that has been incorporated into Laboratory 2 should make it easier for students to conduct "twinning" experiments like those that have such a rich history in developmental biology's past."

** American Society for Microbiology: Cindy L. Munro, "Genetic Technology and Scientific Integrity" (Chapter 10), in Francis L. Macrina (Virginia Commonwealth University), Scientific Integrity (3rd ed.) (American Society for Microbiology Press, March 2005, pf. 259) ( "Two [cloning] techniques (blastomere separation and somatic cell nuclear transfer) are currently available in mammals; blastomere separation has already been demonstrated with human cells. Human cloning is not a new topic for bioethical debate; the U.S. House of Representatives held hearings on the topic in 1978. However, it continues to be a difficult problem for those concerned with scientific integrity, as scientific and technological abilities outpace a consensus regarding appropriate use of the technologies. ... In blastomere separation, a fertilized ovum is developed in vitro to an early multicellular (up to 32-cell) stage. Each of the blastomeres is totipotent at this stage, and careful division of the cell mass yields multiple cell masses, each capable of developing into a genetically identical organism. For example, a 16-cell embryo can be divided to yield two 8-cell masses (resulting in identical twins) or four 4-cell masses (resulting in identical quadruplets). Blastomere separation was first demonstrated with mouse embryos in 1970 and in cattle embryos in 1980. In the context of infertility research, nonviable human embryos were duplicated using this technique in 1993; news reports generated a great deal of public debate regarding the ethics of the technology (18).

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