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Human Cloning and Human Dignity:
An Ethical Inquiry


Table of Contents

The President's Council on Bioethics
Washington, D.C.
July 2002
www.bioethics.gov
Chapter Four

Scientific Background

Introduction

The purpose of this chapter is to provide background on basic scientific aspects of human cloning for readers of this report. Background on stem cell research is also included to enable readers to understand how cloned embryos might be useful in stem cell and other biomedical research. This limited treatment only summarizes and highlights basic aspects of these topics, in part because two major detailed reports, Scientific and Medical Aspects of Human Reproductive Cloning1 and Stem Cells and the Future of Regenerative Medicine,2 have been recently published.

This review is based largely on scientific research papers published through June 2002, supplemented by references to several articles in the popular press. However, the research areas of cloning and stem cell research are being very actively investigated, and significant new developments are published frequently. Publication of new results could change some of the interpretations and emphases in this review.

Use of unfamiliar technical terms has been avoided wherever possible. Scientific names and terms used are described and defined in the Glossary of Terms.

Some Basic Facts about Human Cell Biology and Sexual Reproduction

We begin with some basic facts about human cells, germ cells (egg and sperm), and early embryonic development to provide the background for understanding the mechanism of cloning and the differences between sexual and asexual reproduction.

Normal human cells with nuclei contain forty-six chromosomes, twenty-two pairs plus two X chromosomes if the individual is female, or twenty-two pairs plus one X and one Y chromosome if the individual is male. These chromosomes contain nearly all of the cell's DNA and, therefore, the genes of the cell. During formation of sperm cells, a process of specialized cell division produces mature sperm cells containing twenty-three chromosomes (twenty-two unpaired chromosomes plus either X or Y). During the formation of eggs (oocytes), a process of specialized cell division produces a cell containing two pronuclei, each of which contains twenty-two unpaired chromosomes plus an X. During fertilization, a polar body containing one of these pronuclei is ejected from the egg.

Fusion of egg and sperm cells and the subsequent fusion of their nuclei (the defining acts of all sexual reproduction) produce a zygote that again contains a nucleus with the adult cell complement of forty-six chromosomes, half from each parent [See Figure 1]. The zygote then begins the gradual process of cell division, growth, and differentiation. After four to five days, the developing embryo attains the 100-200 cell (blastocyst) stage. In normal reproduction, the blastocyst implants into the wall of the uterus, where, suitably nourished, it continues the process of coordinated cell, tissue, and organ differentiation that eventually produces the organized, articulated, and integrated whole that is the newborn infant. According to some estimates, about half of all early human embryos fail to implant, and are expelled with the menses during the next menstrual cycle.

Not quite all the DNA of a human cell resides in its nucleus. All human cells, including eggs and sperm, contain small, energy-producing organelles called mitochondria. Mitochondria contain a small piece of DNA that specifies the genetic instructions for making several essential mitochondrial proteins. When additional mitochondria are produced in the cell, the mitochondrial DNA is replicated, and a copy of it is passed along to the new mitochondria that are formed. During fertilization, sperm mitochondria are selectively degraded inside the zygote. Thus, the developing embryo inherits solely or principally mitochondria (and mitochondrial DNA) from the egg.

Human reproduction has also been accomplished with the help of in vitro fertilization (IVF) of eggs by sperm, and the subsequent transfer of one or more early embryos to a woman for gestation and birth. Even though such union of egg and sperm requires laboratory assistance and takes place outside of the body, human reproduction using IVF is still sexual in the biological sense: the new human being arises from two biological parents through the union of egg and sperm.

Egg and sperm cells combined in vitro have also been used to start the process of animal development. Transfer of the resulting blastocysts into the uterus of a female of the appropriate animal species is widely used in animal husbandry with resulting successful live births.

Cloning (Asexual Reproduction) of Mammals

The startling announcement that Dolly the sheep had been produced by cloning3 indicated that it was possible to produce live mammalian offspring via asexual reproduction through cloning with adult donor cell nuclei.i In outline form, the steps used to produce live offspring in the mammalian species that have been cloned so far are:

  1. Obtain an egg cell from a female of a mammalian species.

  2. Remove the nuclear DNA from the egg cell, to produce an enucleated egg.

  3. Insert the nucleus of a donor adult cell into the enucleated egg, to produce a reconstructed egg.

  4. Activate the reconstructed egg with chemicals or electric current, to stimulate the reconstructed egg to commence cell division.

  5. Sustain development of the cloned embryo to a suitable stage in vitro, and then transfer the resulting cloned embryo to the uterus of a female host that has been suitably prepared to receive it.

  6. Bring to live birth a cloned animal that is genetically virtually identical (except for the mitochondrial DNA) to the animal that donated the adult cell nucleus.

Cloning to produce live offspring carries with it several possibilities not available through sexual reproduction. Because the number of presumably identical donor cells is very large, this process could produce a very large number of genetically virtually identical individuals, limited only by the supply of eggs and female animals that could bear the young. In principle, any animal, male or female, newborn or adult, could be cloned, and in any quantity. Because mammalian cells can be frozen and stored for prolonged periods at low temperature and grown again for use as donor cells in cloning, one may even clone individuals who have died. In theory, a clone could be cloned again, on and on, without limit. In mice, such "cloning of clones" has extended out to six generations.4

Diagram of early stages of human fertilization, cloning, and parthenogenesis

Figure 1: Diagram of early stages of human fertilization, cloning, and parthenogenesis.
[Modified from Rick Weiss and Patterson Clark, The Washington Post.]

Since the report of the birth of Dolly the cloned sheep, attempts have been made to clone at least nine other mammalian species. As summarized in Table 1, live offspring have been produced in a low percentage of cloned embryo transfer experiments with sheep, cattle, goats, mice, pigs, cats5 and rabbits.6 According to a press report,7 attempts to clone rats, dogs, and primates using adult cell DNA have not yet yielded live offspring. In experiments to clone different mammalian species, many of the transferred cloned embryos fail to develop normally and abort spontaneously in utero. In addition, a variety of health problems have been reported in many of the cloned animals that survived to live birth.8 However, some surviving cloned cattle appear physiologically similar to their uncloned counterparts, and two cloned cows have given birth to their own offspring.

9,10 Why is production of live cloned mammalian offspring a relatively rare event? Several factors may play a role. Enucleation of the egg may (variably from one attempt to the next) remove or damage its "epigenetic reprogramming" (see Glossary of Terms) capabilities. Isolating a nucleus from the donor cell and manipulating it to insert it into the egg is also a traumatic process that may damage the nucleus. An optimal in vitro nutritive environment for the development of cloned animal embryos may not yet have been determined. One interpretation11 attributes the early death of many cloned embryos to complete failure or incompleteness of epigenetic reprogramming.

Epigenetic Modification and Reprogramming

Normal mammalian embryonic development results from selective expression of some genes and repression of others. Tissue differentiation depends upon several types of "epigenetic modifications" (see Glossary of Terms) of DNA structure and spatial organization that selectively turn genes on and off. The chromosomal DNAs of egg and sperm cells are modified during their maturation, so that at fertilization, both sets of DNA are ready for the complex pattern of gene expression required for normal embryonic development. In order for the DNA of a differentiated adult cell to direct embryonic development in cloning, it must be "epigenetically reprogrammed." That is, the epigenetic modifications that allowed the cell to express genes appropriate for, for example, a differentiated skin cell must be reduced, and the gene expression program required for full embryonic development must be activated.

TABLE 1. SOME COMPARATIVE DATA ON LIVE BIRTHS FROM CLONING OF ANIMALS

[For a more complete collection of data, see the NAS Report Scientific and Medical Aspects of Human Cloning]

Animal Species

Donor Cell

Number of Cloned Embryos Transferred

Number of Live Births

Live Births per Embryo Transfer

Reference

Sheep udder cells (frozen) 29 1 3.4% 1
Cattle fetal fibroblasts
cumullus & oviduct cells
496
10
24-30#
4-8*
4.8-6%
40-80%
2a
2b
Mice cumulus cells 2468 31** 1.3% 3
Goats transgenic fetal fibroblasts
fetal fibroblasts
transgenic fetal fibroblasts
97
85
184
5
3
5
5.2%
3.5%
2.7%
4a
4b
4c
Pigs fetal fibroblasts 110
335
1
5
0.9%
1.5%
5a
5b
Cats cumulus cells 87 1 1.1% 6
Rabbits cumulus cells 371 6 1.6% 7
References:
1. Wilmut, I., et al., Nature, 385: 264-267 (1997)
2a. Cibelli, J.B., et al., Science, 280: 1256-1258 (1998)
2b. Kato, Y., et al., Science, 282: 2095-2098 (1998)
3. Wakayama, T., et al., Nature, 394: 369-374 (1998)
4a. Baguisi, A., et al., Nature Biotechnology, 17: 456-461 (1999)
4b. Keefer, C.L., et al., Biol Reprod, 64: 849-856 (2001)
4c. Reggio, B.C., et al., Biol Reprod, 65: 1528-33 (2001)
5a. Onishi, A., et al., Science, 289: 1188-90 (2000)
5b. Polejaeva, I.A., et al., Nature, 407: 86-90 (2000)
6. Shin, T., et al., Nature, 415: 859 (2002)
7. Chesne, P., et al., Nature Biotechnology, 20: 366-369 (2002)

# Six animals died shortly after birth; * four animals died shortly after birth;
** 20 animals died at a young age [Ogonuki, N.K., et al., Nature Genetics, 30: 253-4 (2002)].

During cloning, cytoplasmic factors in the egg cell reprogram the chromosomal DNA of the somatic cell. In rare cases, this reprogramming is sufficient to enable embryonic development to proceed all the way to the birth of a live animal (for examples, see Table 1). In many cloning experiments, epigenetic reprogramming probably fails or is abnormal, and the developing animal dies. Incomplete epigenetic reprogramming could also explain why some live-born cloned animals suffer from subtle defects that sometimes do not appear for years. 12

The completeness of epigenetic reprogramming is crucial for successful cloning-to-produce-children. It will also be important to assess the impact of variation in epigenetic reprogramming on the biological properties of cloned stem cell preparations. If the extent of epigenetic reprogramming varies from one cloning event to the next, the protein expression pattern and thus the biological properties of cloned stem cell preparations may also vary. Thus, it may be necessary to produce and test multiple cloned stem cell preparations before preparations that are informative about human disease or useful in cellular transplantation therapies can be identified.

Cloning-to-Produce-Children

At this writing, it is uncertain whether anyone has attempted cloning-to-produce-children. Although claims of such attempts have been reported in the press,13,14 no credible evidence of any such experiments has been reported as of June 2002. Thus, it is not yet known whether a transferred cloned human embryo can progress all the way to live birth. However, the steps in such an experiment would probably be similar to those described for animal cloning [see above and references to Table 1]. After a thorough review of the data on animal cloning, the NAS panel, in its report Scientific and Medical Aspects of Human Cloning [page ES-1], came to the following conclusion: "It [cloning-to-produce-children] is dangerous and likely to fail."

Stem Cells and Regenerative Medicine

The subject of stem cell research is much too large to be covered extensively here. Yet the following information on stem cells and their possible uses in medical treatments should facilitate understanding of the relationships between cloning-for-biomedical-research and stem cells (see also the reports Scientific and Medical Aspects of Human Reproductive Cloning and Stem Cells and the Future of Regenerative Medicine).

Stem cells are undifferentiated multipotent precursor cells that are capable both of perpetuating themselves as stem cells and of undergoing differentiation into one or more specialized types of cells (for example, kidney, muscle). Human embryonic stem cells have been isolated from embryos at the blastocyst stage15 or from the germinal tissue of fetuses.16 Multipotent adult progenitor cells have been isolated from sources such as human17 and rodent18 bone marrow. Such cell populations can be differentiated in vitro into a number of different cell types, and thus are the subject of much current research into their possible uses in regenerative medicine. Cloned human embryonic stem cell preparations could be produced using somatic cell nuclear transfer to produce a cloned human embryo, and then taking it apart at the (100-200 cell) blastocyst stage and isolating stem cells (see Figure 2). These stem cells would be genetically virtually identical to cells from the nucleus donor.

Scientists are pursuing the development of therapies based on transplantation of cells for several human diseases, including Parkinson's disease and Type I diabetes. In Parkinson's disease, particular brain cells that produce the essential neurotransmitter dopamine die selectively. Experimental clinical treatment involving transplantation of human fetal brain cell populations, in which a small fraction of the cells produce dopamine, has improved the condition of some Parkinson's disease patients.19 Dopamine-producing neurons derived from mouse embryonic stem cells have been shown to function in an animal model of Parkinson's disease.20 Thus, there is a possibility that transplantation of dopamine-producing neural cells derived from embryonic or adult stem cell populations might be a useful treatment for Parkinson's disease in the future.

However, to be effective as long-term treatments of Parkinson's disease, Type I diabetes, and other diseases, cell transplantation therapies will have to overcome the immune rejection problem. Cells from one person transplanted into the body of another are usually recognized as foreign and killed by the immune system. If cells derived from stem cell preparations are to be broadly useful in transplantation therapies for human diseases, some way or ways around this problem will have to be found. For example, if the cells were isolated from a cloned human embryo at the blastocyst stage, in which the donor nucleus came from a patient with Parkinson's disease, in theory these stem cells would produce the same proteins as the patient. The hope is that dopamine-producing cells derived from these "individualized" stem cell preparations would not be immunologically rejected upon transplantation back into the Parkinson's disease patient. Alternatively, if dopamine-producing cells could be derived from the patient's own adult stem cell or multipotent adult precursor cell populations, they could also be used in such therapies. Another possibility is mentioned in a press report21 about work with a single Parkinson's disease patient, in which brain cells were removed from the patient, expanded by growth in vitro, stimulated to increase dopamine production, and transplanted back into the brain of the same patient with an observed reduction in disease symptoms.

By combining specific gene modification and cloned stem cell procedures, Rideout et al.22 have provided a remarkable example of how some human genetic diseases might someday be treated. Starting with a mouse strain that was deficient in immune system function because of a gene mutation, these investigators (1) produced a cloned stem cell line carrying the gene mutation, (2) specifically repaired the gene mutation in vitro, (3) differentiated the repaired cloned stem cell preparation in vitro into bone marrow precursor cells, and (4) treated the mutant mice with the repaired bone marrow precursor cells and observed a restoration of immune cell function.

Although remarkably successful, the experimental results included a caveat. The investigators also observed a tendency of even these cloned bone marrow precursor cells to be recognized as foreign by the recipient mice. Rideout et al. were led to conclude: "Our results raise the provocative possibility that even genetically matched cells derived by therapeutic cloning may still face barriers to effective transplantation for some disorders."

Lanza et al.23 have also evaluated the potential for immune rejection of cloned embryonic materials, while showing the potential therapeutic value of tissues taken from cloned fetuses. Cloned cattle embryos at the blastocyst stage were transferred to the uteri of surrogate mothers and allowed to develop for five to eight weeks. Fetal heart, kidney, and skeletal muscle tissues were isolated, and degradable polymer vehicles containing these cloned cells were then transplanted back into the animals that donated the nuclei for cloning. The investigators observed no rejection reaction to the transplanted cloned cells using two different immunological tests. More investigations with cloned stem cell materials involving different stem cell preparations of varying sizes, different sites of implantation, and sensitive tests to detect low levels of immunological rejection will be required for a complete assessment of the possibility of using cloned stem cell populations to solve the immune rejection problem.

Stages in the development, isolation, and transformation of embryonic stem cells.

Figure 2: Stages in the development, isolation, and transformation of embryonic stem cells.
[Modified from Rick Weiss and Patterson Clark, The Washington Post.]


Human Cloning-for-Biomedical-Research

Producing cloned stem cell preparations for possible use in individual patients suffering from diseases like Parkinson's disease and Type I diabetes is one reason to pursue cloning-for-biomedical-research.24 In vitro production of cloned human embryos could also be important to scientists interested in studying early human development. Stem cells derived from cloned human embryos at the blastocyst stage that were produced with nuclei from individuals with genetic diseases could be useful in the study of the critical events that lead to these diseases (for example, see Bahn et al.25 ). Specific genes could be introduced into developing human embryos to obtain information about the role or roles of these genes in early human development.

One attempt at human cloning-for-biomedical-research has been published in the scientific literature by Cibelli et al.26 as of the end of June 2002.ii It involved the following steps (see Figure 1):

  1. Obtain human eggs from informed and consenting female volunteers.

  2. Remove the nuclear DNA from the egg cell, to produce an enucleated egg.

  3. Insert the nucleus of a cell from an informed and consenting adult donor into the enucleated egg, to produce a reconstituted egg.

  4. Activate the reconstituted egg with chemicals or electricity to stimulate it to commence cell division in vitro, producing a cloned embryo.

  5. Use a microscope to follow the early cell divisions of the cloned embryo.

In the experiments described by Cibelli et al., the stated intent was to create cloned human embryos that would progress to the 100-200 cell stage, at which point the cloned embryo would be taken apart, stem cells would be isolated from the inner cell mass, and an attempt would be made to grow and preserve "individualized" human stem cells (see Figure 2) for the possible future medical benefit of the somatic cell donor. Because the cloned human embryos stopped dividing and died at the six-cell stage, no stem cells were isolated in these experiments. In light of results in other animal species and the variable completeness of "epigenetic reprogramming," it is perhaps not surprising that sixteen of the nineteen cloned human embryos described by Cibelli et. al. did not undergo cell division and none of the other three divided beyond the six-cell stage.

Although the steps these researchers followed in these experiments were the same as those that would be used by those attempting human cloning-to-produce-children, they distinguished their intent from such cloning by stating: "Strict guidelines for the conduct of this research have been established by Advanced Cell Technology's independent Ethics Advisory Board (EAB). In order to prevent any possibility of reproductive cloning, the EAB requires careful accounting of all eggs and embryos used in the research. No embryo created by means of NT [nuclear transfer] technology may be maintained beyond 14 days of development."

Parthenogenesis (Another Form of Asexual Reproduction)

Using chemical or electrical stimuli, it is also possible to stimulate human eggs to undergo several rounds of cell division, as if they had been fertilized (see Figure 1). In this case, the egg retains all forty-six egg cell chromosomes and egg cell mitochondria. In amphibians, this asexual reproduction process, known as parthenogenesis, has produced live offspring that contain the same nuclear DNA as the egg. These offspring are all necessarily female. Parthenogenesis in mammals has not led reproducibly to the production of live offspring. 27

Cibelli et al.26 activated human eggs (obtained from informed and consenting donors) by parthenogenesis, and obtained multiple cell divisions up to the early embryo stage in six out of twenty-two attempts. Although there was no report that stem cells were isolated in these experiments, it is possible that parthenogenesis of human eggs could induce them to develop to a stage where parthenogenetic stem cells could be isolated. For example, Cibelli et al.28 derived a monkey parthenogenetic stem cell preparation from Macaca fasicularis eggs activated by parthenogenesis. Whether cloned stem cells resulting from parthenogenesis have been completely and correctly epigenetically reprogrammed remains to be determined.



ENDNOTES

  1. National Academy of Sciences (NAS). Scientific and Medical Aspects of Human Reproductive Cloning, Washing-ton, DC. National Academy Press, 2002.
  2. National Research Council/Institute of Medicine (NRC/IOM). Stem Cells and the Future of Regenerative Medicine. Washington DC. National Academy Press, 2001.
  3. Wilmut, I., et al. "Viable offspring derived from fetal and adult mammalian cells" Nature, 385: 810-813, 1997.
  4. Wakayama, T., et al. "Cloning of mice to six generations" Nature, 407: 318-319, 2000.
  5. Shin, T., et al. "A cat cloned by nuclear transplantation" Nature, 415: 859, 2002.
  6. Chesne, P., et al. "Cloned rabbits produced by nuclear transfer from adult somatic cells" Nature Biotechnology, 20: 366-369, 2002.
  7. Kolata, G. "In Cloning, Failure Far Exceeds Success" New York Times, December 11, 2001, page D1.
  8. See Table 2 in Reference 1.
  9. Lanza, R.P., et al. "Cloned cattle can be healthy and normal," Science, 294: 1893-1894, 2001.
  10. Cibelli, J.B., et al. "The health profile of cloned animals" Nature Biotechnology, 20: 13-14, 2002.
  11. Rideout III, W.M., et al. "Nuclear cloning and epigenetic reprogramming of the genome" Science, 293: 1093-1098, 2001.
  12. Ogonuki, N., et al. "Early death of mice cloned from somatic cells" Nature Genetics, 30: 253-254, 2002.
  13. Weiss, R. "Human Cloning Bid Stirs Experts' Anger; Problems in Animal Cases Noted" The Washington Post, April 11, 2001, page A1.
  14. Brown, D. "Human Clone's Birth Predicted; Delivery Outside U.S. May Come By 2003, Researcher Says" The Washington Post, May 16, 2002, p. A8.
  15. Thomson, J.A., et al. "Embryonic stem cell lines derived from human blastocysts" Science, 282: 1145-1147, 1998.
  16. Shamblott, M.J., et al. "Derivation of pluripotent stem cells from cultured human primordial germ cells" Proc Nat Acad Sci U.S.A., 95: 13726-13731, 1998.
  17. Reyes, M., et al. "Origin of endothelial progenitors in human postnatal bone marrow" Journal of Clinical Investigation, 109: 337-346, 2002.
  18. Jiang, Y., et al. "Pluripotency of mesenchymal stem cells derived from adult marrow," Nature, 418: 41-49, 2002.
  19. Hagell, P., and P. Brundin, "Cell survival and clinical outcome following intrastriatal transplantation in Parkinson's disease" J Neuropathol Exp Neurol, 60: 741-752, 2001.
  20. Kim, J-H., et al. "Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson's disease" Nature 418: 50-56, 2002.
  21. Weiss, R. "Stem Cell Transplant Works in Calif. Case, Parkinson's Traits Largely Disappear," The Washington Post, April 9, 2002, p. A8.
  22. Rideout III, W.M., et al. "Correction of a genetic defect by nuclear transplantation and combined cell and gene therapy" Cell, 109: 17-27, 2002.
  23. Lanza, R.P., et al. "Generation of histocompatible tissues using nuclear transplantation" Nature Biotechnology, 20: 689-696, 2002.
  24. Lanza, R.P., et al. "Human therapeutic cloning" Nature Medicine, 5: 975-977, 1999.
  25. Bahn, S., et al. "Neuronal target genes of the neuron-restrictive silencer factor in neurospheres derived from fetuses with Down's syndrome: A gene expression study" The Lancet, 359: 310-315, 2002.
  26. Cibelli, J.B., et al. "Somatic cell nuclear transfer in humans: Pronuclear and early embryonic development" ebiomed: The Journal of Regenerative Medicine, 2: 25-31, 2001.
  27. Rougier, N., and Z. Werb. "Minireview: Parthenogenesis in mammals" Mol Reprod Devel 59: 468-474, 2001.
  28. Cibelli, J.B., et al. "Parthenogenetic stem cells in nonhuman primates" Science, 295: 819, 2002.

_______________

  1. Previous experiments dating from the 1950s had shown that it was possible to clone amphibians. Earlier experiments had also produced clones of animals using embryonic donor cells. What made the report of Dolly's birth stand out was the fact that a mammal had been cloned, and from cells taken from an adult.
  2. According to a press report (Hall, C.T., "UCSF Admits Human Clone Research: Work to Duplicate Embryos for Medical Purposes on Hold" San Francisco Chronicle, May 25, 2002, p. A1), other attempts to produce cloned human embryos for biomedical research were carried out at the University of California-San Francisco by Roger Pedersen and coworkers in 1999 and 2001. Another press report (Leggett K., and A., Regalado "China Stem Cell Research Surges as Western Nations Ponder Ethics," Wall Street Journal, March 6, 2002, p. A1) indicates that as-yet-unpublished work in China by Sheng Huizhen involved insertion of human somatic cell nuclei into enucleated rabbit eggs, and that the resulting cloned embryos developed to a stage where human embryonic stem cells could be isolated.



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