Let's Be Clear About the Science and Ethics of iPS Cell Research and Its Reproductive Possibilities

B. Current Genetic Engineering Techniques That Turn "Cells" Into New Embryos for Reproductive Purposes

One of the genetic engineering techniques for reproductive cloning that the UNESCO document identified was "tetraploid complementation. Examples of the use of tetraploid complementation are documented in the studies that follow.

1. Triploid and Tetraploid Complementation

It is commonly, but mistakenly, thought that "pluripotent" stem cell can't develop into new embryos, even though they do have the capacity to eventually develop into the three major human tissues of an adult. That is because, e.g., such "stem cells" lack the ability to make the outer layer of cells that normal embryos have which eventually form the placenta, umbilical cord, etc.

But what if you take a "pluripotent" stem cell - human embryonic (HE) or reprogrammed iPS cell -- insert it into or fuse it with an artificial "shell" that will function as and substitute for an embryo's outer layer of cells, and then implant it as you would any normal embryo? That is what has recently been accomplished, and it works. It is now possible to genetically engineer such new embryos for reproductive purposes by means of "tetraploid complementation" (TC) which provides a substitute for the outer layer of cells of an embryo that is normally responsible for producing the later placenta, umbilical cord, etc.

"Tetraploid complementation" started off as an "assay" used to test a pluripotent stem cell's "potency" - the ability of a stem cell to differentiate into the three germ layers and tissues of an adult human being. It has now also become a scientific method of reproducing new embryos per se. There are already several variations on the theme. For example, according to NIH:

Scientists studying mouse chimeras (mixing cells of two different animals) noted that fusing two 8-cell embryos produces cells with 4 sets of chromosomes (tetraploid cells) that are biased toward developing into extra-embryonic tissues such as the placenta. The tetraploid cells do not generate the embryo itself; the embryo proper develops from injected diploid stem cells. This tendency has been exploited to test the potency of a stem cell. Scientists begin with a tetraploid embryo. Next, they inject the stem cells to be tested. If the injected cells are pluripotent, then an embryo develops. If no embryo develops, or if the resultant embryo cannot survive until birth, the scientists conclude that the cells were not truly pluripotent.19

Thus what is involved here is that in tetraploid complementation the cells of two mammalian embryos can be combined to form a new multi-ploid embryo. The technique is now used to construct genetically modified organisms, to study the consequences of certain mutations on embryonic development, and in the study of pluripotent stem cells.

To explain further, the tetraploid complement assay can start with producing a tetraploid embryo in which every chromosome exists fourfold. This is done by taking an embryo at the two-cell stage and fusing the two cells of the embryo by applying an electrical current. The resulting tetraploid embryo will continue to divide, and all daughter cells will also be tetraploid. Such a tetraploid embryo can develop normally to the blastocyst stage and will implant into the wall of the uterus. The tetraploid cells can form the extra-embryonic tissue (placenta etc.), but a proper fetus will rarely develop. However, one can now combine such a tetraploid embryo (either at the morula or blastocyst stage) with normal diploid embryonic stem cells (ES) or iPS cells from a different organism. The resulting embryo will then develop normally. The fetus is exclusively derived from the ES cell, while the extra-embryonic tissues are exclusively derived from the tetraploid cells.

Foreign genes or mutations can also be introduced into ES or iPS cells rather easily, and these ES and iPS cells can then be grown into whole animals using the tetraploid complementation "assay". Also, by introducing targeted mutations into the tetraploid cells and/or into the ES or iPS cells, one can study which genes are important for embryonic and fetal development and which ones are important for development of the extra-embryonic tissues. Finally, tetraploid complementation is also used to test whether induced pluripotent stem cells (stem cells artificially produced from differentiated cells, e.g. from skin cells) are as competent as normal embryonic stem cells, or if a viable animal can be produced from an induced pluripotent stem cell using the tetraploid complementation.

** Thus in Nagy et al's 1973 article, ""Derivation of completely cell culture-derived mice from early-passage embryonic stem cells", it was demonstrated that several newly generated mouse embryonic stem (ES) cell lines were able to produce completely ES cell-derived mice by using tetraploid complementation.20

** In 2003, Tam and Rossant's study, "Mouse embryonic chimeras: tools for studying mammalian development", also documented that, "By combining embryonic stem cell technology, molecularly tagged mutations and sensitive cell lineage markers, chimeras can provide invaluable insights into the tissue-specific requirement and the mode of action of many mouse genes."21

** Lan Kang et al confirm in their 2009 report, "iPS cells can support full-term development of tetraploid blastocyst-complemented embryos", that, "This work serves as a proof of principle that iPSCs can in fact generate full-term embryos by tetraploid complementation."22

** Also in 2009 Xiao-yang Zhao et al confirm in their study, "iPS cells produce viable mice through tetraploid complementation" that IPS cells produce viable mice through tetraploid complementation.23

** Naumann describes in his report, ""Production Of tetraploid mouse embryos by electrofusion", the use of tetraploid complementation as a means of producing new embryos that are chimeras (have genomes from different species). For example, combining tetraploid blastocysts with either embryonic stem (ES) cells or diploid embryos has become an established technique for creating mouse chimeras. In both methods the tetraploid cells are not able to contribute to the embryo itself, but instead create the primitive endoderm derivatives and the trophectoderm. He also explains how complex phenotypes can be produced by generating embryos carrying mutations of multiple genes.24

** Bertolotti praises these techniques in his study, "Fully pluripotent iPS cells: Mouse tetraploid complementation, ethical human ES-like cells and reproductive cloning ban". However, he begrudgingly acknowledges some concerns: "Such a genesis of fertile adult "iPS" mice could revive the roguish temptation of banned human reproductive cloning currently doomed by the failure of human cell reprogramming by somatic cell nuclear transfer (SCNT)."25

** Denker also acknowledges concerns in his study, "Induced pluripotent stem cells: how to deal with the developmental potential", but seems to indicate that it is not a problem:

"Recent developments in research on embryonic stem cells and induced pluripotent stem cells suggest that potentiality of cells should be a new focus in stem cell research ethics and policy. Successful reconstitution of viable embryos from induced pluripotent stem cells using tetraploid complementation has been reported and indicates a way for direct cloning of individuals from these cells. This together with recent observations on gastrulation and pattern formation processes in cultures of embryonic stem cells has considerable ethical relevance after the advent and worldwide spread of induced pluripotent stem cell technology. Available knowledge of the molecular basis of mammalian embryology now makes it possible to envisage ways to deal technically with the ethical dilemma of stem cell potentiality."26

On the other hand, some have tried to alert the public about the ethical and scientific implications of producing new embryos by means of tetraploid complementation.

** For example, in 2009 the Chinese researchers Shi V. Liu, Ke-Ming Cui, and Wei-Kang Zhang attempted to voice their concerns by letter to the journals Nature and Science. The publication of both letters was denied, so these researchers published them elsewhere as "Open Letters".

In their first letter to Nature, "Why Do We Need "Fully Pluripotent" iPS Cells?",27 these researchers questioned the scientific and ethical value of obtaining "fully pluripotent" iPSCs (induced pluripotent stem cells) "using an embryo-sacrificing reproductive cloning technique involving tetraploid complementation, as presented already in some studies published in Nature. Scientifically. For example, they referred to such research as "over-reprogramming" and "over-extension of iPS technology", questioning why researchers should rush into reproductive cloning using some incorrectly programmed stem cells or "man-made cancer stem cells", and pointing out that studies have shown that iPS cells are already more aggressively tumorgenic than HECS's. The researchers also noted that such research would violate NIH Guidelines for funding. Further, they cautioned against following "an extreme point of regenerative medicine" that thinks it ideal to find a "fully pluripotent" stem cell so that a damaged or aged body can be regenerated completely from just one such cell of the same genetic identity - calling such "ideals" a form of "extremely selfish immortality". They were also concerned about the effect that such "extremes" would have on the reputation of good researchers, as well as concerned that once this success in non-human animals is translated into human studies, these dangerous techniques would be applied to humans in clinical trials or abused as biological weapons.

In their second letter to Science, "iPS mice: Passing acid test or breaking basic ethics?",28 the researchers reiterated many of the same concerns as in their letter to Nature. They also took issue with the whole concept of tetraploid complementation as "the gold standard" or "acid test" for "full pluripotency", i.e., determining whether a "pluripotent" cell is capable of "forming an entire viable organism. Quite interestingly, they point out one of the common points of erroneous scientific disinformation long used in most of this research, i.e., the insistence that embryonic cells at the very early two-cell stage of an embryo, would not develop into any embryos on their own, that they were only "pluripotent". (Such a claim directly contradicts the research identified by UNESCO as "embryo splitting", and that is addressed later in this article.)

The researchers point out that "it is an enigma why a "full pluripotency" cell would even require two "disabled" embryos to support their "full" development. It is also puzzling why successful complementation from "full pluripotency" cells often yields only less than 100% chimaerism." They suggest that the "gold standard" is not 100% gold, asking why a non-destructive and simple and transparent "gold standard" - implanting noting but just the testing cell(s) - has not been used for testing the "full pluripotency" of iPSCs:

Ethically, it is difficult to imagine that a method requiring destruction of at least two embryos per standard or original complementation assay would be considered as more "ethical" than a method such as SCNT which utilizes just one egg per nuclear transfer. Considering an egg is just half of a potential future life, we simply cannot accept the fact that damaging two living embryos for generating a potential life is any more ethical practice, regardless whether or not the cells contributing the "full pluripotency" are ethical or not. If iPSCs are already ethical cells aimed for therapeutic cloning, please do not pass them through an unethical reproductive cloning to demonstrate their "full pluripotency" which is not even needed for normal partial body regenerative medicine. We should not destroy more whole lives for obtaining some parts for treating diseases or extending lifespan!29

They conclude by asking, "why should we jump over this mice embryo-killing 'success' in passing an 'acid test' which, proved in principle, basic ethics can be easily broken intentionally or accidentally".30

** Indeed, others have also recently reaffirmed the increased tendency of iPS cells to form tumors, as Tong et al describe in their study, "Mice generated from tetraploid complementation competent iPS cells show similar developmental features as those from ES cells but are prone to tumorigenesis". As the authors noted, "Previously, we and others have demonstrated that fully reprogrammed iPSCs were capable of producing full-term mice via the tetraploid complementation method, yet a thorough postnatal development evaluation of iPS mice is still lacking."31

** Another verification of the tumorgenicity of iPS cells comes from Shi-Lung Lin in his article, "Concise Review: Deciphering the Mechanism Behind Induced Pluripotent Stem Cell Generation". Hopefully, regenerative medicine using pluripotent or multipotent stem cells holds a great promise in developing therapies for treating developmental abnormalities, degenerative disorders, and aging-related illness. However, supply and safety of the stem cells are two major problems with today's regenerative medicine. Recent development of induced pluripotent stem cells (iPSCs) has overcome the supply shortages by allowing the reprogramming of patients' body cells to embryonic stem cell (ESC)-like pluripotent cells. Still, the potential tumorigenicity of iPSCs remains as an obstacle.32

** David Cyranoski also expressed serious concerns about the mice studies using iPS cells and tetraploid complementation to reproduce whole new embryos. In his 2009 article, "Mice made from induced stem cells: Technical feat shows that the different route to stem cells can indeed make a full mammal body",33 Cyranoski states the obvious, that the recent studies do answer " a lingering question about the developmental potential of the cells." Since Shinya Yamanaka of Kyoto University in Japan created the first iPS cells in 2006, "researchers have wondered whether they could generate an entire mammalian body from iPS cells, as they have from true embryonic stem cells. ... Experiments reported online this week suggest that, at least for mice, the answer is yes." And he also cautions that this would essentially be a new way to clone adult mammals  reprogramming DNA from an adult and generating a genetically identical individual. As a potentially easier method that produces fewer abnormalities than conventional cloning, he cautions that "it might evoke interest among mavericks as a tool for human cloning."

** Some secular bioethicists have also voiced concern, writing a lengthy and detailed report that includes several charts of relevant legislation within the United States as well as internationally. The point of their article is best summed up with their statement: "The use of iPSCs and tetraploid complementation for human reproductive cloning would raise profound ethical objections. Professional standards and laws that ban human reproductive cloning by somatic cell nuclear transfer should be revised to also forbid it by other methods, such as iPSCs via tetraploid complementation."34 (emphases added)

** More recently Andrew Pollack again cautions on the use of genetic engineering techniques to derive stem cells for therapeutic use with sick patients, and their application to reproductive cloning: "But the potential problem with iPS cells might make some scientists take another look at making patient-specific tissues by creating an embryo from a patient's cell through so-called therapeutic cloning. That approach ... is controversial because the same technique might also be used to create a baby."35

2. Artificial Sex Gametes Derived From iPS, Human Embryonic, and Adult Cells

One might even take any human cell, deprogram it back to a primitive germ cell, or an iPS cell, allow those cells to mature into sperm and oocytes, then use them in fertilization to reproduce a new embryo for reproductive purposes.

a) Documentation 2000 - 2008

The Hinxton Group has been tracking and documenting research in which any human cell can be deprogrammed back to a primitive germs cell for quite a while now. To see their extensive listing of what is termed "pluirpotent stem cell-derived gametes" (PSCDG) research for the years 2000 - 2008, see this endnote summary.36 More recently, there has been an acceleration of such research.

b) PSCDG Documentation 2008 - 2011

** There are already several books on the use of stem cells and tetraploid complementation for human reproductive purposes. Bongso and Lee describe many of them in their book, Stem Cells: From Bench to Bedside.37 Interestingly, by the time the book was published much of the research involving the production of human sperm and oocytes from HE and iPS cells was already accomplished. But one of the articles in that book by Burley constitutes a good link between the use of these techniques to reproduce human embryos for reproductive purposes and their use to derive human sex gametes from adult cells.

As Burley writes in his contribution to the book:

iPS cells have been combined with a tetraploid embryo (the fusion of two cultured blastomeres) to produce baby mice that are genetically identical to the iPS cells used. This raises the specter of human reproductive cloning in a new guise, assuming that the technique of tetraploid complementation could be replicated using human cells. In addition, as noted above, it is predicted that iPS technology may one day enable researchers to produced gametes from pluripotent stem cells. Two methods have been suggested: the combination of iPS-derived male and female gametes, and the combination of an iPS-derived gamete, such as a sperm with an egg of in vivo natural origin. ... iPS technology could allow the production of artificial gametes from the cells of living or dead people (frozen tissue in tissue banks) of known identity. When the implications of artificial gamete creation are more fully appreciated, it is inevitable that some will argue that it should not be allowed at all because too much scope for abuse exists. ... Artificial gametes would be very easy to make from a person's cells without his/her knowledge, and could be used to create an embryo for research or an embryo for implantation. These worries are, I think, well founded. However, there is no hard and fast causal relationship between a technology and all of its possible uses.38

** In 2008, the editor of Science noted in his article, "Sperm cells created from female embryo", that British scientists, headed by Prof. Nayernia, who had already coaxed human male bone marrow cells to develop into primitive sperm cells, had just repeated the feat with human female embryonic stem cells. As the editor wrote, "Sperm cells have been created from a female human embryo in a remarkable breakthrough that suggests it may be possible for lesbian couples to have their own biological children." The University of Newcastle team that had achieved the feat was applying for permission to turn the bone marrow of a woman into sperm. The editor noted that the research raises the possibility of lesbian couples one day having children who share both their genes as sperm created from the bone marrow of one woman could be used to fertilize an egg from her partner. Prof Nayernia showed the potential of the method in 2006, when he used sperm derived from male embryonic stem cells to fertilize mice to produce seven pups, six of which lived to adulthood, though the survivors did suffer problems.39

** In 2009, researchers from Spain and Australia published a review study, "Differentiation of germ cells and gametes from stem cells".40 Marques-Mari et al note that advances in stem cell research have opened new perspectives for regenerative and reproductive medicine, and that several groups have reported their ability to differentiate stem cells into germ line cells. Some of them have been successful in obtaining male and female gamete-like cells by using different methodologies. Marque-Mari et al highlighted studies reporting development of germ cell-like cells from murine and human embryonic (ESC) and somatic stem cells They noted that published studies indicate that germ cells can be consistently differentiated from mouse and human ESC, "although certain differentiation obstacles remain to be resolved". Their conclusion was that differentiation of germ cells from both iPS and HE stem cells "has the potential of becoming a future source of gametes for research use, and if genetic and epigenetic methodological limitations could be solved, therapeutic opportunities could be also considered."41

** In 2009, Amander Clark, a developmental biologist at the University of California, Los Angeles, and her research team created human egg and sperm precursors using an existing line of human induced pluripotent stem (iPS) cells. "For couples who can't seem to get pregnant, one of the more common causes is egg or sperm quality", the article noted.42 Researchers had previously shown that embryonic stem cells could produce egg- and sperm-cell precursors. But infertile couples would need to use donor eggs or sperm obtained from infertility clinics. "The benefit of using a human iPS cell is that it has the donor's own genetics," Clark says. The study also highlights the differences between embryonic stem cells and iPS cells. When Clark compared the developmental potential of iPS cells to that of embryonic stem cells, she found that the latter resulted in egg and sperm precursors that were substantially healthier, with fewer chromosomal abnormalities.

** Another focus on "infertility" treatments involved a study concerned with male infertility. In 2009, Neri et al pointed out in their study, "Treatment options for impaired spermatogenesis: germ cell transplantation and stem-cell based therapy", that men diagnosed with germ cell aplasia can only be treated by donor or de novo generated human gametes. In the past several years there have been attempts to manufacture gametes by inducing haploidization of somatic cells and more recently, by generating sperm-like cells through embryonic stem cell differentiation.43

** In early 2011 a French journal published another review study concerning "infertility" treatments.44 The article by Assou et al, "Generation of artificial gametes from human pluripotent stem cells: Is this possible?", noted how both human embryonic stem cells and iPS stem cells are both "pluripotent", and therefore "they have remarkable properties of self-renewal and differentiation into the three cellular lineages, somatic as well as germinal". The article also notes that the formation of putative gametes from mouse pluripotent stem cells has been shown, the apparent derivation of functional mouse male gametes has also been described, and that "in vitro production of [human] gametes by differentiation of human iPS has become a very immerging aspect of the actual research constituting an alternative tool for infertility treatment."

** Recently, a bioethical panel at Japan's Keio University in Japan announced its approval for research to create human reproductive cells (sperm and ova) using iPS cells to treat infertility and other congenital diseases.45 Prof. Hideyuki Okano of the university's School of Medicine and the Tokyo-based Kato Ladies Clinic will lead the research. The research team will try to transform human iPS cells into human reproductive ones by injecting reagents into human stem cells created from human skin cells.

** Another review study, "Application of iPS in assisted reproductive technology: sperm from somatic cells?", was published in September 2011 by Yao and his research team.46 Noting that "infertility is an emotionally charged problem, and it is a condition of increasing incidence", they present a hypothesis for a cure for infertile men with non-obstructive azoospermia (NOA) (caused by testis failure or impaired spermatogenesis). They suggest that "induced pluripotent stem (iPS) cell-derived spermatozoa can be a potential source for male gametes for patients with NOA",. They cite the evidence already established: the generation of viable, live-born, fertile mature organism from iPS cells; the successful induced differentiation of male gametes from pluripotent cells in vitro; and the wide use of intracytoplasmic sperm injection (ICSI) in human assisted reproduction.

** Finally, in November 2011 a Federal Government-appointed panel in Australia has recommended the use of the controversial "in vitro-derived gametes" in its final report to the review of national cloning and embryo research laws, "opening the door for a woman to produce a baby by creating her own sperm from skin cells."47 The technology - if proven - "could allow same-sex couples, or those who cannot produce their own eggs or sperm, to have genetically related children from both parents." It could also wipe out genetically linked diseases from one partner, and allow a man to develop his own eggs to fertilize his sperm in a surrogate. The panel points out that scientists have proven they can produce mice from natural egg and artificial sperm, and Britain's national fertility and embryology authority estimates eggs and sperm could be developed within five years to be potentially used in infertility treatment. Though the proposed legislation bans using artificial gametes for reproduction for now, bringing Australia in line with Britain, the report warns the community that the "development should be anticipated".

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