11.2Cloning and Stem Cell Technology

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Cloned Sheep

Fig. 11-4. Cloned Sheep "Dolly" (front) and its Surrogate Mother Sheep
Photo courtesy of , The Roslin Institute, The University of Edinburgh

On February 23, 1997, the Sunday issue of English newspapers reported the news of the birth of the cloned sheep "Dolly." Dolly had already been born at the Roslin Institute in Edinburgh in July 1996. But her birth was not disclosed to the public, and it was supposed to be published in the issue of the scientific journal Nature of February 27, 1997. One may assume that somehow the newspaper got ahead in the game and scooped the news. However, in actuality, the journal Nature had circulated the official release date for major media beforehand, but the newspaper broke it. With this event, one could imagine how big the news of Dolly's birth was at the time. The birth of the cloned sheep Dolly started off with a mammary cell of a 6-year-old female sheep. The nucleus of this mammary cell was transferred into an enucleated sheep egg, and a cell resembling a fertilized egg was created. Then this cell was placed into the uterus of a surrogate mother sheep, resulting in the birth of Dolly (Fig. 11-4, Fig. 11-5).

Fig. 11-5. Creation of a Cloned Sheep

Fertilized eggs are "pluripotent" and can develop into any kind of cell, tissue, or organ. The astonishing fact about Dolly's case is that it defied the commonly accepted biological theory, "that mammalian cells, which develop from a fertilized egg, determine their roles through differentiation (see Chapter 5), and these cells cannot return to being omnipotent such as fertilized eggs." Furthermore, this technology can produce a new organism with genetic information completely identical to one already grown up.
The birth of Dolly immediately stirred recalled worldwide debate on the possibility of the birth of a "human clone."
In Japan, in response to this situation, first, the Council for Science and Technology Policy (at that time) imposed a moratorium on human cloning and set up a bioethics committee. In January 1998, as its subordinate organization, the Cloning Subcommittee of the Bioethics Committee began to operate, and in December 1999, the subcommittee's report was acknowledged. Based on this report, the Bill Draft on the Regulation of Cloning Technologies (officially called Law on the Regulation of Cloning Technologies Relating to Human proposal) was submitted to the Diet and approved in December 2000. This law prohibited human cloning and banned the development of special embryos by cloning techniques, and regulated their transplantation into the uterus (see Column below).


Law on Cloning and Special Guidelines on Embryos

The Law on Cloning describes the total of nine particular embryos, including cloned human embryos, and defines how they should be handled.
A "human-animal clone embryo" is an embryo generated by a process in which the nucleus of a human somatic cell is transplanted into an animal egg cell from which the nucleus was removed. A "human split embryo" is an embryo in which the human embryo is split up. A "human embryonic nuclear transfer embryo" is an embryo into which the nucleus of split embryonic cells is transferred. A "human-human chimeric embryo" is an embryo with which different human cells are mixed. "Human-animal chimeric embryos" and "animal-human chimeric embryos" are embryos in which human and animal cells are mixed. A "human-animal hybrid embryo" is an embryo derived from the fertilization of a human egg and an animal sperm, or the fertilization of an animal egg and a human sperm. An "animal-human clone embryo" is an embryo generated by the process in which one removes the nucleus of a human egg and transfers it into an animal cell.
This law does not prohibit these nine specified embryos from being produced. However, it bans the transfer of the following 4 types of embryos into the uterus: "human somatic cell nuclear transfer embryos (cloned human embryos);" "human-animal hybrid embryos;" "human-animal clone embryos;" and "human-animal chimeric embryos." Those who violate the law will be imposed "a maximum of 10 years of imprisonment or fined a maximum of 10,000,000 yen."
With regard to the transfer of the remaining five types of embryos into the uterus, and the production and research of all specified embryo types, one is obliged to notify the Minister of Education, Culture, Sports, Science and Technology, according to the Guidelines on the Handling of Specified Embryos. These guidelines prohibit the transfer of all specified embryos into the uterus and production and research of the eight types of embryos, except for "animal-human chimeric embryos." However, in 2003, the government-run Council for Science and Technology Policy laid out a policy to accept the production and research of cloned human embryos.

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Human ES Cells

Fig. 11-6. Creation of an ES Cell

In November 1998, when the debate on the regulation of cloning technologies was in progress, yet another important technology relating to the generation of human beings was developed—the establishment of human embryonic stem cells (human ES cells).
ES cells can be created by growing fertilized animal eggs up to the "blastocyt" stage, at which clumps inside the cells are removed, and subsequently, cells are cultivated under certain conditions (Fig. 11-6). Before the arrival of ES cells, mouse stem cells had already been produced and had been used to create chimeric and knockout mice (see Fig. 11-2). From the mice, it was found that the main characteristics of ES cells are: "they are pluripotent and can differentiate into any kind of cell" and "they can be cultivated for a long period of time in an undifferentiated state (namely without the role of the cell being determined)". The term "stem cell" means cells with various growth properties, similar to branches growing out of the stem of a tree.
Through the establishment of human ES cells, a strong interest in the use of ES cells for "regenerative medicine" (see Chapter 5) has risen. This is because there were expectations that the pluripotency of ES cells would enable cells, tissues, and organs that have been harmed or lost through illness or injury to be replicated. One example is to grow certain neurocytes that are lacking in the brain of a patient with Parkinson's disease with ES cells and transplant them. Another is to transplant neurocytes produced from ES cells into a person who is paralyzed due to a spinal cord injury. Moreover, there were further expectations that producing organs such as heart and liver in vitro would be made possible in some distant future.
On another note, for the creation of human ES cells, it is necessary to destroy the embryo that has been raised from a fertilized egg, and this has caused ethical debate in various countries (see Column below).


Regulation of Human ES Cells and Cloned Embryos in Various Countries

The ethics of production of human ES cells was raised, particularly in countries with a Christian background such as Europe and the United States. Conservative Christian groups such as Catholics believe that the life of a human begins from the moment when an egg is fertilized. With this idea, to them, the destruction of fertilized eggs for the production of ES cells is equal to murder.
What is more important? The preservation of fertilized eggs to protect life? Or the possibility to save a patient by regenerative medicine? The balance between these two questions varies from person to person and from country to country.
In the United States, ES cells even became a focal point in the presidential election. The Bush administration of the Republican Party, which was based on the support of religious conservatives, prohibited the contribution of federal funds for the production of human ES cells. In response, scientists and the House demanded that research on ES cell be promoted, which created a conflict in the U.S. Congress. However, the Democratic Party thinks different about this issue, and it is believed that with a change of the ruling administration government policies on human ES cells will change.
In England, under certain conditions, both the production of human ES cells and cloned human embryos are permitted. In Germany, through the Embyro Protection Law, neither the production of human ES cells nor the production of cloned human embryos is allowed. In France, under certain conditions, the production of human ES cells is permitted, but the production of cloned human embryos is prohibited. The United States does not permit public funds to be used for the production of human ES cells or cloned human embryos.

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Japanese Guidelines on Human ES Cells

In Japan as well, there was debate on human ES cells, which eventually led to the formation of the country's guidelines in September 2001 that permit the production of human ES cells under strict conditions. The guidelines permit the production of ES cells only for the purpose of research, and not for the clinical application of ES cells, considering that human embryos are "the sprout of human life," and stipulating that "they must be handled in spirits of sincerity and caution."
A fertilized egg used to produce human ES cells is limited to a "surplus embryo." The surplus embryo, which is among cryogenically preserved embryos produced from a couple undergoing fertility treatment for assisted reproduction technologies, loses its role for treatment (see Column Section 7 of Chapter 5). Research institutions which have the permission to establish are obliged to comply with specific conditions regarding their research performance, technical competence, etc. Medical institutions that provide fertilized eggs and research institutes that produce ES cells are required to undergo double assessments from an in-house ethics committee and national review committee.
These strict conditions reflect the general awareness that human embryos are not recognized as human life itself, but that they must be handled differently than ordinary cells.

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Cloned Human Embryos

As mentioned above, the Guidelines on the Handling of Specific Embryos under the Act on the Regulation of Human Cloning Techniques forbid the production of cloned human embryos. However, in Japan as well, there were disputes regarding the production of such embryos at the Bioethics Expert Committee, which was set up by the Council for Science and Technology Policy of the Cabinet Office. Two groups of opposite views debated over the issue: one group believed that the production of cloned human embryos was indispensable for the realization of regenerative medicine, whereas the other believed that the production of such embryos was premature and that ethical issues were also still unclear. The group of pro human embryo cloning advocates that "such embryos are conducive to the realization of regenerative medicine that causes no adverse reactions." Even if ES cells produced from fertilized eggs could be used to create cells or tissues for therapeutic use, there is still a possibility that this would cause adverse reactions in the patient. However, if such cloned embryos were produced from somatic cells of the patient, from which ES cells can be produced, then it could possibly prevent adverse reactions.
On the other hand, the group of people who are either cautious or against the production of cloned human embryos think as follows: the question of whether or not ES cells produced from cloned embryos can be used for regenerative medicine has not been fully answered even at the animal experiment level. Cloned human embryos have a potential to become cloned human beings when they are placed in the uterus and allowed to grow in it, and thus, a thoughtless production of such embryos is unacceptable. In addition, the production of cloned human embryos requires large amounts of human eggs, and the ethical issues involved herein have not yet been solved.
In contrast, the people in favor of the use of such embryos counter that "there are also things that are still not clear even after the animal experiment." Eventually, in July 2004, both groups reached a conclusion that allowed production of cloned human embryos by imposing certain conditions on handling of eggs, protection of women, etc.
Regarding the production of ES cells with cloned human embryos, one incident occurred in South Korea. It was discovered that research papers had been fabricated (see Column below) and there were some ethically inappropriate cases involved in the handling of eggs during research.
How can we balance the possibility of their use for medical care and ethical issues involved in the handling of eggs and cloning techniques? Such debate continues over the question of at what stage one should shift from the animal experiment to experiments involving humans.


The Human ES Cell Hoax

In 2004, a research group led by Professor Hwang Woo-suk at Seoul University published a research paper that they had produced ES cells from cloned human embryos. In 2005, they published another paper that they had produced ES cells from cloned human embryos derived from cells of a patient with spinal cord injury. These reports attracted attention worldwide as achievements that could take us closer to the realization of regenerative medicine. However, from 2005 to 2006, it was discovered that these results had been fabricated. This incident also allowed us to take a closer look at the ethical norms of scientists in a fight for research results.
Professor Hwang Woo-suk's group received more than 2000 eggs for their research and paid compensation for those eggs, although as a rule they should be free of charge. In January 2005, South Korea passed the Bioethics Law to introduce international standards into its cutting-edge life sciences research. Yet, they found a gap between the sense of ethics in their fields of research and Western ethical norms.

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iPS Cells

In a pursuit of overcoming the weaknesses of human ES cells and cloned human embryos, researchers have gone to great lengths to produce cells equal to ES cells using neither eggs nor fertilized eggs.
In 2007, a research group at Kyoto University succeeded in producing cells with pluripotency similar to ES cells by introducing four different genes into human skin cells. These cells are called induced pluripotent stem cells (iPS cells). With iPS cells, it is no longer necessary to destroy human fertilized eggs or to produce cloned human embryos, and pluripotent cells can be produced from the cells of the patient. Today, iPS cells receive much attention in medical research, since these cells may lead to the production of reproductive medicine without adverse reactions. However, there is a risk that iPS cells may develop into cancer cells, and their safety has not been established yet. No techniques to enable stable production of various cells and tissues for regenerative medicine have been realized. Moreover, there is a possibility that cells such as eggs and sperm can be produced from iPS cells; therefore, it is vital to set the rules in place.

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Somatic Stem Cells

ES and iPS cells are pluripotent and are able to differentiate into various kinds of cells. Among somatic cells, which have differentiated to a certain degree, certain cells exist that can differentiate into various kinds of cells. Such cells are called somatic stem cells (see Chapter 5).
Somatic stem cells include: hematopoietic stem cells, which differentiate into white blood cells, red blood cells, etc; neural stem cells, which differentiate into various nerve cells; and mesenchymal stem cells, which differentiate into bones, cartilage, heart muscle, etc. There are opinions that these kinds of cells are more practical and easy to apply because we can avoid the ethical issues involved with fertilized eggs and cloned human embryos. On the other hand, there are also opinions that there is a limit to the types of the cells that can differentiate. As such, simple comparisons with ES and iPS cells are not easy.

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