Parthenogenetic Human Stem Cells

I. Parthenogenesis

Parthenogenesis is the creation of an embryo without fertilizing the egg with a sperm, thus omitting the sperm’s genetic contributions. To achieve this feat, scientists “trick” the egg into believing it is fertilized, so that it will begin todivide and form a blastocyst.

A. Definition

The fact that in certain animals, females may produce eggs capable of development without previous copulation has been knownfor a long time and was called 'lucina sine concubitu'. Richard Owen (1849) coined the term ‘parthenogenesis’ and who defined it as procreation without the immediate influence of a male'; this includes various processes such as fission and budding in addition to the development of unimpregnated ova (Owen, 1849). Several authors have since attempted to redefine the term.

According to Suomalainen (1950), parthenogenesis means 'the development of the egg cell into a new individual without fertilization', but this simple definition is adequate for many lower animals. In other animals, however, embryos produced without fertilisation suffer a high mortality, a fact that is reflected in the definition of parthenogenesis by Beatty as 'the production of an embryo from a female gamete without the concurrence of a male gamete, and with or without eventual development into an adult'.5 He then subsequently modified this definition by substituting 'without any genetic contribution froma male gamete' for 'concurrence of a male gamete' (Beatty, 1967).The purpose of the extending of the definition was to include thespecial case of gynogenesis, in which a spermatozoon enters an egg and activates it to complete the second meiotic division and to develop into an embryo, but does not contribute any

chromosomal material. Whether or not this process should be included within the province of parthenogenesis is clearly a matter of choice.

It will be clear however, that parthenogenesis does not necessarily imply that the mother has had no access to a male.

In different groups of animals both fertilised and unfertilised eggs of the same female may proceed to form embryos.Parthenogenesis in plants has been discussed by Stebbins (1950) and by Nygren (1954). The term 'apomixis' is commonly used with reference to plants and here modes of reproduction other than parthenogenesis may be included (Suomalainen, 1950).

On any definition, however, parthenogenesis is distinct fromasexual reproduction since it involves the production of egg cells, whereas in asexual reproduction new individuals are formedfrom somatic cells of the parent. (Mittwoch, 1987)

B. Types of Parthenogenesis

Parthenogenesis is divided further into two. It is divided based on its cytological processes.

1. Apomictic Parthenogenesis

Parthenogenesis can occur without meiosis through mitotic oogenesis. Development of a new individual in the absence of the fusion of male and female gametes is known as apomixis. The term,however, has been defined in different ways by different authors.According to Beatty (1957), apomixis in animals is synonymous with parthenogenesis, whereas White (1977) restricts the term to parthenogenesis in the complete absence of meiosis. The term 'apomixis' is plants differs from that in animals, since in plants a diploid sporophyte generation alternates with a haploid gametophyte generation, and so the breakdown of fertilisation mayhave different consequences (Mittwoch,1987)

2. Automictic Parthenogenesis

Parthenogenesis involving meitotic processes is called Automictic parthenogenesis. In automictic parthenogenesis, meiosis takes place but the chromosal constitution of the mother is restored through one or several different mechanisms. Some of these mechanisms enforce homozygozity at all loci while other mechanisms pass the genome of the mother intact to the offspring

(Mittwoch, 1987).

These mechanisms are the following: (a) premeiotic replication of the chromosomes followed by a normal meiosis; (b) normal meiosis followed by the fusion of the first or second cleavage nuclei; (c) abnormal meiosis I followed by a normal meiosis I1 where kinetochore replication occurs; (d) normal meiosis I with meiosis I1 aberrant, or equivalently the second polar body fuses with the egg nucleus; (e) abnormal meiosis I andI1 in which kinetochores replicate and two of the four chromatidsare randomly distributed between a single polar body and the egg nucleus; and (f) inhibition of meiosis I with restoration of zygoidy by utilization of meiosis II products as the first cleavage nuclei (Asher, 1970)

II. Occurrence of Parthenogenesis

Parthenogenesis could either be natural or artificial. As what was said, parthenogenesis occurs naturally within plants, some invertebrates and few in vertebrates especially within fish,amphibians, reptiles and rarely in birds. In plants, parthenogenesis is usually termed with apomixis. However in animals, it usually occurs within the lower order.

In the artificial occurrence, induced parthenogenesis is done. The experimental induction of parthenogenesis in mammals began with the pioneering studies of Pincus and his collaboratorsin the rabbit. In 1936 Pincus and Enzman showed that the

extrusion of polar bodies could be induced in vitro not only by contact with sperm suspension, but also by heat treatment or exposure to butyric acid and hypertonic solutions. Subsequently Pincus and Shapiro (1940) described the effect of cold treatment on unfertilised tubal eggs in vitro and claimed not only an increased incidence of cleavage but also the production of a living young. There has since been abundant confirmation of the possibility of inducing parthenogenetic development in mammals byexperimental procedures but none of the embryos so formed has survived the embryonic period (Mittwoch, 1978).

A. Recorded Study of Parthenogenesis in Humans

There was no clear record of a study of parthenogenesis in humans because it may happen naturally yet remained unnoticed. Parthenogenetic reproduction could occur among human females yetremain unnoticed. Indeed, such a woman could have a husband and be totally unaware of her own condition. She would have only daughters, each of which would carry only her genes, which would almost certainly increase in the gene pool, at least over the short term. Is there any evidence for this? Claims of reproduction without males are not to be expected from nunneries,but neither have any emanated from prisons where women are kept isolated from men.

Parthenogenesis in humans may seem far-fetched, but 50 yearsago no-one suspected that parthenogenesis could occur in any vertebrate: now all-female species have been documented in fish, amphibians, reptiles and birds (all major orders of vertebrates except mammals). A study was done but yet there was no clear conclusion (Pianka, 2014)

But on June 26, 2007, International Stem Cell Corporation (ISCC), a California-based stem cell research company, announced that their lead scientist, Dr. Elena Revazova, and her research team were the first to intentionally create human stem cells from

unfertilized human eggs using parthenogenesis. The process may offer a way for creating stem cells that are genetically matched to a particular woman for the treatment of degenerative diseases that might affect her. In December 2007, Dr. Revazova and ISCC published an article illustrating a breakthrough in the use of parthenogenesis to produce human stem cells that are homozygous in the HLA region of DNA. These stem cells are called HLA homozygous parthenogenetic human stem cells (hpSC-Hhom) and have unique characteristics that would allow derivatives of these cells to be implanted into millions of people without immune rejection. With proper selection of oocyte donors according to HLA haplotype, it is possible to generate a bank of cell lines whose tissue derivatives, collectively, could be MHC-matched witha significant number of individuals within the human population (Revazova et al., 2007).

On August 2, 2007, after much independent investigation, it was revealed that discredited South Korean scientist Hwang Woo-Suk unknowingly produced the first human embryos resulting from parthenogenesis. Initially, Hwang claimed he and his team had extracted stem cells from cloned human embryos, a result later found to be fabricated. Further examination of the chromosomes ofthese cells show indicators of parthenogenesis in those extractedstem cells, similar to those found in the mice created by Tokyo scientists in 2004. Although Hwang deceived the world about beingthe first to create artificially cloned human embryos, he did contribute a major breakthrough to stem cell research by creatinghuman embryos using parthenogenesis. The truth was discovered in 2007, long after the embryos were created by him and his team in February 2004. This made Hwang the first, unknowingly, to successfully perform the process of parthenogenesis to create a human embryo and, ultimately, a human parthenogenetic stem cell line.

III. Stem Cell

A. Definition

The term stem cell includes a large class of cells defined by their ability to give rise to various mature progeny while maintaining the capacity to self-renew. Embryonic stem cells (ESCs) were first isolated from the inner mass of late blastocysts in mice by Sir Martin J. Evans and Matthew Kaufman (Evans & Kaufman, 1981) and independently by Gail R. Martin (Martin, 1981). Later, it became possible to obtain ESCs from non-human primates and humans. In 1998, James Thomson and his team reported the first successful derivation of human ESC lines (Thomson et al., 1998), thus extending the great potential of ESCs by providing the opportunity to develop stem cell-based therapies for human disease.

hES cells are derived from the so‐called inner cell mass ofblastocyst stage embryos that develop in culture within 5 days offertilization of the oocyte (Thomson et al., 1998; Reubinoff et al., 2000). Although hES cells can form all somatic tissues, theycannot form all of the other extraembryonic’ tissues necessary for complete development, such as the placenta and membranes, so that they cannot give rise to a complete new individual. They aretherefore distinct from the totipotent fertilized oocyte and blastomere cells deriving from the first cleavage divisions. hES cells are also immortal, expressing high levels of a gene called telomerase, the protein product of which ensures that the telomere ends of the chromosomes are retained at each cell division and the cells do not undergo senescence. The only other cells with proven pluripotency similar to that of ES cells are embryonic germ (EG) cells, which as their name implies, have beenderived from ‘primordial germ cells’ that would ultimately form the gametes if the fetus had not been aborted. In humans, hEG cells were first established in culture in 1998, shortly after the first hES cells, from tissue derived from an aborted fetus

(Shamblott et al., 1998). Biologically, hEG cells have many properties in common with hES cells (Shamblott et al., 2001).

In the adult individual, a variety of tissues have also beenfound to harbour stem cell populations. Examples include the brain, skeletal muscle, bone marrow and umbilical cord blood, although the heart, by contrast, contains no stem cells after birth (reviewed in McKay 1997; Fuchs and Segre, 2000; Watt and Hogan, 2000; Weissman et al., 2000; Blau et al., 2001; Spradling et al., 2001). These adult stem cells have generally been regarded as having the capacity to form only the cell types of the organ in which they are found, but recently they have been shown to exhibit an unexpected versatility (Ferrari et al., 1998;Bjornson et al., 1999; Petersen et al., 1999; Pittenger et al., 1999; Brazelton et al., 2000; Clarke et al., 2000; Galli et al., 2000; Lagasse et al., 2000; Mezey et al., 2000; Sanchez, Ramos etal., 2000; Anderson et al., 2001; Jackson et al., 2001; Orlic et al., 2001). Evidence is strongest in animal experiments, but is increasing in humans, that adult stem cells originating in one germ layer can form a variety of other derivatives of the same germ layer (e.g. bone marrow to muscle within the mesodermal lineage), as well as transdifferentiate to derivatives of other germ layers (e.g. bone marrowtobrain between the mesodermal and ectodermal lineages). To what extent transdifferentiated cells are immortal or acquire appropriate function in host tissue remains largely to be established but advances in this area are rapid, particularly for multipotent adult progenitor cells (MAPCs) of bone marrow (Reyes and Verfaillie, 2001). Answers to these questions with respect to MAPCs, in particular whether theyrepresent biological equivalents to hES and can likewise be expanded indefinitely whilst retaining their differentiation potential, are currently being addressed (Jiang et al. 2002; Schwartz et al., 2002; Verfaillie, 2002; Zhao et al., 2002). For other adult stem cell types, such as those from brain, skin or intestine (Fuchs and Segre, 2000), this may remain unclear for

the immediate future. Although the discussion here concerns hES cells and the use of embryos, the scientific state of the art on other types of stem cell is important in the context of the subsidiarity principle.

B. Possibility of Human Embryo Development

In creating human stem cells through parthenogenesis, an unfertilized (haploid) oocyte is treated chemically such that it becomes diploid, with two identical sets of the maternal chromosomes. These uniparental embryos are by definition gynogenetic and never result in viable offspring, because they fail to generate extra embryonic tissues. Nevertheless, in mice (Boediono et al.,1999) and in apes (Cibelli et al.,2002), parthenotes have been shown to develop to the blastocyst stage and yield cell lines with properties not distinguishable from ES cells derived from fertilized oocytes. However, in view of the fact that some genes are genomically imprinted, such that they are expressed only if inherited via the male germ line, ES cells derived from parthenotes may well be abnormal. First attempts at parthenogenesis in humans have not yielded hES cell lines (Cibelli et al., 2002). It is important to realize that such hES cell lines, if developed in humans, would only provide a tissue match for the oocyte donor, i.e. women of reproductive age. Although it has been speculated that two sets of male chromosomescould also be used in parthenotes, there is no evidence that thisis a real option.

Just like embryonic stem cells, parthenogenetic stem cells can be coaxed to grow into different kinds of human cells or tissue, ready to be transplanted into diseased areas of the body.International Stem Cell scientists have converted them into livercells and plan to convert them into neurons for treating Parkinson’s disease, pancreatic cells for diabetes, and other tissues. Meanwhile teams at the Massachusetts-based Bedford Stem

Cell Research Foundation are working to improve the efficiency ofmethods of deriving stem cells from parthenotes.

Normal mammalian development requires genomic contributions from both the mother and the father. Mammalian parthenotes do notsurvive to term, because the maternal and paternal chromosomes ofmammals are not equivalent (McGrath and Solter, 1984). Uniparental duplications of regions of some chromosomes are lethal or detrimental to the embryo. Eleven such regions have been found (Searle and Beechey, 1978); in a few of these regions,genes have been identified that are expressed from only one allele, either maternal or paternal. For the embryo to distinguish between the two alleles, at some point in gametogenesis the genes must be marked as maternal or paternal. This gametic imprint causes a functional difference in the gene product, such as transcription of the gene (Barlow,1994). The genes that have been identified as imprinted have a wide range offunctions, from splicing factors, such as Snrpn and (potentially)Sp2, to growth factors, such as insulin (Ins1 and Ins2) and Igf-2, to genes that are functional as RNAs, such as H-19 and Xist (reviewed by Bartolomei, 1994).

Gametic imprinting, presumably, is the main cause of uniparental failure in development, although nonimprinted genes, such as insulin-like growth factor-1 receptor (Igf-1r), may be misregulated in parthenogenetic embryos (Rappolee et al., 1992). Parthenogenetic embryos fail in development in a characteristic fashion. The most advanced parthenogenetic embryos survive to theearly limb bud stage. These embryos have little extraembryonic tissue and almost no trophoblast (Kaufman et al., 1977). Most parthenogenetic embryos develop into a disorganized mass of parietal endoderm (PE) cells (Sturm et al., 1994). Although supplying parthenogenetic stem cells (Allen et al., 1994) or embryos (Spindle, A., Sturm, K., Flannery, M., Meneses, J., Wu, K. and Pedersen, R., unpublished data) with trophoblast allows a

higher percentage of the embryos to reach the early limb bud stage, the embryos still die, showing that both genomes are needed for normal embryonic development. Studies with chimeras show that both genomes are needed in at least some cells of the embryo. In chimeras between normal (zygotic) embryos and parthenotes, parthenogenetic cells are excluded from day 6.5 p.c.trophoblast, but not from the inner cell mass (ICM) derivatives in the embryo proper (Clark et al., 1993). At midgestation, parthenogenetic cells are excluded from parts of the embryo proper, including skeletal muscle, liver and pancreas (Fundele etal., 1990). Therefore, parthenotes fail not only because they develop a small trophoblast, but also because of some cell-autonomous defects.

Pincus (1939) demonstrated parthenogenetic activation of mammalian eggs using temperature and chemical stimuli. Thus far, parthenogenetic activation of eggs has been studied in a variety of mammals including mice, goats, cows, monkeys, and humans. Plachot et al. described parthenogenesis in humans by examining 800 human oocytes and showed that 12 activated parthenogenetically and four underwent normal cleavage. Although there have been no reports of naturally-occurring human parthenotes, a human parthenogenetic chimera has been described .The juvenile patient presented with developmental delay, apparent sex reversal, and entirely parthenogenetic blood leukocytes. This finding confirmed the viability of chimeras in higher mammals as presaged by successful murine experiments over the previous two decades.

C. Somatic Cell Nuclear Transfer (Therapeutic Cloning)

Before discussing the ethical issues around ‘therapeutic cloning’, the term itself requires consideration. To avoid confusion, it has been proposed that the term ‘cloning’ be reserved for reproductive cloning and that ‘Nuclear transplantation to produce stem cells’ would be better

terminology for therapeutic cloning (NAS report, 2002; Vogelsteinet al., 2002). Others have pointed out the disadvantage of this alternative term, namely that it masks the fact that an embryo iscreated for instrumental use. More important in our opinion however, is that the use of the adverb ‘therapeutic’ suggests that hES cell therapy is already a reality: strictu sensu there can only be a question of therapeutic applications once clinical trials have started. In the phase before clinical trials, it is only reasonable to refer to research on nuclear transfer as ‘research cloning’ or ‘nuclear transplantation for fundamental scientific research’, aimed at future applications of therapeuticcloning.

Some consider this technology to be ethically neutral; they claim that the ‘construct’ produced is not a (preimplantation) embryo. Qualifications suggested for these constructs include: activated oocyte, ovasome, transnuclear oocyte cell, etc. (Kiessling, 2001; Hansen, 2002) However, to restrict the definition of ‘embryo’ to the product of fertilization in the post Dolly era is a misleading anachronism. Although the purpose of therapeutic cloning is not the creation of a new individual and it is unlikely that the viability of the constructed product is equivalent to that of an embryo derived from sexual reproduction, it is not correct to say that an embryo has not been created.

The core of the problem is that here, human embryos are created solely for instrumental use. Whether or not this can be morally justified—and if so, under what conditions—has already been an issue of debate for years in the context of the development of ‘assisted reproductive technologies’ (ART).

IV. Bioethics

Stem cell research offers great promise for understanding basic mechanisms of human development and differentiation, as

well as the hope for new treatments for diseases such as diabetes, spinal cord injury, Parkinson’s disease, and myocardialinfarction. However, human stem cell (hSC) research also raises sharp ethical and political controversies. The derivation of pluripotent stem cell lines from oocytes and embryos is fraught with disputes about the onset of human personhood. The reprogramming of somatic cells to produce induced pluripotent stem cells avoids the ethical problems specific to embryonic stemcell research. In any hSC research, however, difficult dilemmas arise regarding sensitive downstream research, consent to donate materials for hSC research, early clinical trials of hSC therapies, and oversight of hSC research. These ethical and policy issues need to be discussed along with scientific challenges to ensure that stem cell research is carried out in anethically appropriate manner. This article provides a critical analysis of these issues and how they are addressed in current policies.

A. Embryonic Stem Cell Research and the Religious Fanctions

Many of the world’s religious leaders have opined about the moral acceptability of stem cell research. While research using adult or cord-blood stem cells is relatively uncontroversial, there is no consensus on the acceptability of using human embryosin research of any kind and therefore, no consensus on the acceptability of deriving human embryonic stem cell (hES) lines or using those cell

lines in research. There is also no consensus on whether somatic cell nuclear transfer (SCNT) should be used to create hES lines with specific characteristics. Religious duties to 1) respect andprotect human life because it is sacred, and 2) to prevent and alleviate human suffering, create tension in evaluating the moralacceptabilityof hES research from a religious perspective. A third religious duty to protect and promote distributive justice

by providing access to therapies for all people creates boundaries or limits on acceptable stem cell research.

Articulated religious opinions about stem cell research primarily deal with the moral acceptability of hES research, based on whether blastocysts are viewed as persons. In moral philosophy a distinction is made between that which is human life, as opposed to nonhuman life or non living material, and human persons. Human persons enjoy a special moral status, and a right to life goes with that status. As such, others have a duty not to harm them. Human life, on the other hand, while mandating respect does not equate with personhood and all the rights to protection that go with it. Some religions accept this distinction while others do not. For some religions, human embryos are human persons from the moment of conception, while for others the important moment is the moment of ensoulment, whenthe embryo or fetus develops or obtains a soul. Ensoulment is sometimes thought to occur weeks or months after conception. These differing opinions reflect diverse views held by and withinthe major world religions. It is important to note that without exception, groups within each of the major religions hold contrary views on the morality of hES research (Knowles, 2000)

B. Embryonic Stem Cell Research and the Society

There is more to the hES cell story than chapters covering scientific themes. These cells are derived from early human embryos that, for many people, carry moral status. Like many new technologies, successful development and use of the cells for human therapeutics will depend not only on their safety and efficacy, but also on their acceptability to society at large. Although Geron’s Ethics Advisory Board (Lebacqz 1999), the National Bioethics Advisory Commission (1999), and the American Association for the Advancement of Science (Chapman 1999)

published suggested guidelines for ethical development of therapies based on these cells, the debate is not over. Modern societies have the obligation to choose which alternative technologies they wish to support to improve their lives. Our hope at Geron is that after a thorough examination of the issues,many of which are explored in this volume, most people will support continued development of the technology, as do patient advocacy groups, bioethics boards, and the medical and scientificcommunities generally. 3e

IV. Conclusion

Stem cells have the capacity for prolonged self-renewal and can produce at least one type of highly differentiated or specialized descendant (Watt and Hogan 2000; Weissman 2000). The hES cells are important because they have certain critical characteristics. Most important they are pluripotent—they are able to develop into many types of tissues (thus, they are also sometimes called pluripotent stem cells, or PSCs). They are also immortal—able to continue dividing indefinitely without losing their genetic structure. Moreover,hES cells are malleable—they can be manipulated without losing cell function. Indeed, studies with animal stem cells suggest that they can be moved into another blastocyst and it will continue its development. Finally,they express the enzyme telomerase, which allows cells to grow and divide.

With the breakthroughs of Dr. Revazova and Scientist Hwang Woo-Suk, stem cells from unfertilized human oocytes have been converted, grown and used to treat several diseases. With this, it is also coined alongside the therapeutic cloning, wherein the

nucleus is removed and then replaced by the nucleus from a somatic cell obtain from the patient.

In spite of this importance—and perhaps because of it—hES cell research has proved to be one of the most controversial developments of the last decades. Controversy arises for many reasons. The research touches deep questions about the nature of human life, limits of interventions into human cells and tissues,and the meaning of our corporate existence. We have probably onlybegun to identify and approach the important ethical issues. However, the immediate controversy revolved around a cluster of difficult and sometimes seemingly intractable questions.

Even with the infinite potentials of curing several diseases by the stem cells created by the unfertilized human oocytes, the ethical and moral implications of the research and therapy have yet to be determined. The debate being, mostly, influenced by the religious beliefs of the citizens which are objecting the research and therapy’s nature but some are approving on some grounds. With the experts and citizens, they are still arguing whether the research and therapy is acceptable to be used and is ethical in nature.

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