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Microsoft Word - mf08001.docABSTRACT
Mammalian embryo biotechnology with multiple applications in reproductive and therapeutic medicine has been summarized in this review article. Risk factors at the molecular, genetic and chromosomal level that profoundly influence the outcome of reproductive cloning have been included for serious considerations. Interspecies embryo cloning has turned out to be a unique bioassay to reveal the embryonic developmental capacities of various adult somatic cells. New approaches for in-vitro cloning of individual blastomeres (early embryonic cells) will lead to ES cells without destroying embryos. Novel attempts for embryo splitting have been presented with various implications for assisted reproduction. Predictive progress and prognostic views for human embryo biotechnology and patients' benefits are outlined for the near future. Social and ethical issues concerning embryo biotechnology are being discussed in reference to public opinion polls or ethics committee guidelines. Key Words: somatic cell nuclear transfer (SCNT), interspecies embryo cloning, embryo splitting, blastomere cloning, assisted reproductive technologies (ART) Embryo biotechnology in reproduction
Primate embryo cloning via somatic cell nuclear transfer (SCNT) with adult donor cells has been carried out by American researchers who reported on the failure to obtain pregnancies from rhesus monkey cloned embryos. From their observations they concluded that “primate NT (nuclear transfer) appears to be challenged by stricter molecular requirements for mitotic spindle assembly than in other mammals” (1). However, there is no further detailed information on biological or species-specific factors responsible for these negative findings on rhesus monkey cloning.
Whether complex embryo biotechnology (Figure 1) will be successfully applied in human
Correspondence: Prof. Dr. Karl Illmensee, Genesis Fertility Center, Via Filopoimenos 24, Patras 262 21 , Greece. Tel.: (30) 2610 270 166, Fax: (30) 2610 221 401, e-mail: [email protected]
reproductive medicine depends on scientific progress and social acceptance (2). In 2001, an opinion poll among medical practitioners and members of APART (international association of assisted reproductive technology centers) has revealed that three quarters of them would be willing to provide human cloning for patients in clinically indicated cases (3). Human reproductive cloning has sparked worldwide disputes for and against its application in medicine (4,5). Some crucial questions concerning reproductive cloning have already been answered in various animal systems (6,7) but other imperfections linked to reproductive SCNT remain to be solved (8). Some of the central questions are focusing on ethical issues and the impact on society. With regard to medical indication, reproductive cloning may be envisaged for infertile couples with a male partner totally devoid of any germ cells. For these couples, it could be highly desirable to conceive a child with the genes of at least one partner, if they have personal or religious reservations about using
Middle East Fertility Society Journal Vol. 13, No. 1, 2008 Copyright © Middle East Fertility Society
Vol. 13, No. 1, 2008 Illmensee Embryo biotechnology in reproductive medicine 1
Figure 1. Biotechnology on mammalian embryos at various stages of preimplantation development (from oocyte to blastocyst) with
multiple applications in veterinary and human reproductive medicine.
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donor gametes. In such cases, “reproductive SCNT would meet an infertile couple’s desire to participate biologically in the development of a new human being”, as stated in the Ethics Committee Report of the American Society for Reproductive Medicine (9). Other important questions relevant to reproductive SCNT are closely linked to yet unsolved risk factors in cloning procedures. I also share proper respects to these biological and medical concerns. A currently negative position on possible applications of reproductive SCNT in humans is intimately connected to the limiting cloning results obtained in farm animals. It has been proposed that further insights into the biological mechanisms and improvement of SCNT technologies are required as essential prerequisites (10).
The first intrauterine transfer of a cloned human embryo has recently been accomplished and employed for an infertile couple although no pregnancy was established in this case (11). In the future, transfer of human cloned embryos may eventually be applicable for patients that have no other alternative options for procreating their own offspring. Currently, however, it should clearly be emphasized that human embryo cloning for reproductive purposes is unsafe from a scientific and medical point of view and is burdened by sincere ethical considerations and severe objections not only expressed by the scientific community but also by public opinion (12). Risk factors in embryo biotechnology
Several limiting factors for reproductive
cloning have been discussed and proposed for intensive investigations (13). Not only epigenetic alterations in methylation of genes, but also changes in structure of chromosomes can be envisaged as critical (14). Multiple factors involved in DNA methylation, chromatin remodelling, genomic imprinting or telomere-size alteration of chromosomal ends can influence profoundly nuclear reprogramming from adult to embryonic gene expression and therefore affects the outcome of animal cloning (6). In the sheep system, it was shown that established cell lines from cloned sheep exposed the same telomere size as the original cell lines used for SCNT cloning
(15). In the cattle system, on the other hand, rebuilding of chromosomal telomeres was documented in cloned calves (16,17). Japanese researchers have reported that remarkable differences in telomere lengths can be observed among cloned cattle derived from different donor cell lines (18). Further cloning experiments on this subject of telomeric extension by exposing donor cell nuclei to the telomerase pool of recipient oocytes are therefore desirable to advance in our knowledge about aging and rejuvenating processes in cultured SCNT donor cells and cloned animals.
Some of the key investigations have focused on nucleocytoplasmic interactions and reprogramming of the transferred somatic cell nucleus by the cytoplasm of the recipient oocyte (19), chromatin remodelling of the somatic nucleus by oocyte factors (20) or cell-cycle coordination between nucleus and cytoplasm during the cloning procedure (21). Cloning efficiency may be further improved by increasing biological uniformity between recipient oocytes and donor cells and by establishing selection techniques and bioassay model systems for functional reprogramming of the adult donor cell genome (22,23). Interspecies bioassay for embryo cloning and stem cells
For future applications in reproductive cloning
it will be very important to further advance in our understanding how to rejuvenate and reprogram human adult cells by modern bioengineering. In this context, we have recently started a novel approach using adult human ovarian granulosa cells and skin fibroblasts for SCNT into enucleated bovine oocytes (24). The rational for selecting these adult cell types was based on various cloning reports in different mammalian species that these cells seem to be currently the most efficient adult donor cells for SCNT procedures (25, 26, 27). Such bioassay could be useful to examine the developmental potential of various human adult cells for their embryonic reprogramming. We documented that SCNT procedures using enucleated bovine oocytes fused with adult human cells can yield appreciable success rates of embryo development up to the blastocyst stage (Figure 2). These interspecies embryos were analyzed
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Figure 2. Interspecies embryo cloning via SCNT. Enucleation of mature bovine oocyte. The polar body and metaphase-II complex are removed from the oocyte. Adult human donor cell is injected into the perivitelline space between enucleated oocyte and zona
pellucida. Interspecies embryos at various stages of preimplantation (from cleavage, morula to blastocyst). individually for the presence of human genomic DNA, human and bovine mtDNA using species- specific microsatellite markers for genomic DNA and species-specific primers for mtDNA. The PCR data revealed that human genomic DNA specific for the corresponding donor cell genome was present in the interspecies embryos. Furthermore, human mtDNA together with bovine mtDNA was detected which clearly proves that mitochondria from the human donor cells were carried over to the enucleated bovine oocytes (24). The interspecies embryos can therefore be considered as heteroplasmic for mtDNA. In our bovine-human SCNT, human-specific mtDNA was still detectable up to the blastocyst stage, similar to what has been reported for interspecies blastocysts derived from rabbit-monkey SCNT (28). In another study on bovine-human SCNT, human-specific mtDNA
could only be detected up to the morula stage (29). Such variability in the persistence of donor cell- specific mtDNA in interspecies embryos needs further investigations. Possible mixing and recombination of different mtDNA populations in cloned animals have been discussed in the context of assisted reproduction (30).
Chinese researchers have reported on interspecies SCNT using enucleated rabbit oocytes fused with human adult fibroblast cells. From the developing cloned embryos they established in- vitro cultures of interspecies-derived ES cells. These ES cells could also differentiate into various tissue- specific cells (31). They also documented by DNA genotyping and cytogenetic karyotyping that the SCNT embryos and ES cells had originated from the genomic DNA of the human fibroblast donor cells. The promising perspectives
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of such an approach are that interspecies embryos may serve as potential source for establishing stem cells. Extensive investigations will be required to discover the embryonic capacity of human adult somatic cells in interspecies SCNT and their ability to create ES cells to be tested for their differentiation potential (32). As important advantage, no human oocytes are required for SCNT in interspecies embryo biotechnology. This crucial issue of utilizing human oocytes in research is heavily criticized or even illegal in several countries worldwide. With regard to these considerations, very recently in England, interspecies embryo cloning for stem cell research has been legalized by British law.
Embryonic stem cells for future therapeutic applications
Australian researchers have first managed to
successfully culture ES cells from human preimplantation embryos (33). ES cells are derived from the inner cell mass (ICM) of cultured human blastocysts produced by in-vitro fertilization for clinical purposes in assisted reproduction (ART) and donated by patients. ES cells remain diploid and maintain their embryonic and proliferative characteristics over many cell divisions in culture. Cellular bioengineering on ES cells will help tremendously to provide particular precursor cells for specific organ therapy (34).
Human embryo-derived ES cells may be employed for future therapeutic purposes since they are capable of differentiating into a variety of tissues and possess a large regenerative potential (35). First therapeutic applications have recently been attempted by using human ES cells for treatment of patients with degenerative diseases (36,37). Human ES cell therapy can be made applicable to repair damaged heart tissue (38). In the future, patients suffering from Parkinson’s disease (39) or diabetes (40) may also be treated successfully with human ES cells. Therapeutic treatments with human ES cells should also be envisaged for patients with pulmonary diseases (41). The extraction and expansion of human ES cells capable of tissue- specific differentiation will open up novel and quite controversial applications for therapeutic medicine (42). Another promising
alternative for creating stem cells has recently been proposed in the context of reprogramming somatic cells into stem cells (43). Such an approach would bypass ethical issues concerning the use and destruction of human embryos for the derivation of stem cells. Embryo cloning for human embryonic stem cells
American researchers have reported on human
SCNT in the context of future therapeutic cloning (44). Using skin fibroblasts and ovarian cumulus cells as nuclear donor types and human enucleated oocytes as SCNT recipients, they obtained two abnormal embryos. At this early embryonic stage, however, it is not yet feasible to establish ES cells which are usually obtained from the ICM of blastocysts. Recently, American researchers have published very promising results on human SCNT using adult fibroblasts and donated oocytes for cloning procedures. Development of closed blastocysts was confirmed at the molecular level by genomic and mitochondrial DNA analysis (45). In another report using oocytes donated from IVF patients and cultured ES cells for SCNT, one cloned human blastocyst developed and was shown to be genomically identical with the ES donor cells, as proven by PCR and DNA analysis. However, attempts to create ES cells from this blastocyst failed in this study (46). With respect to human therapeutic cloning by means of developing cloned embryos in assisted reproduction, a novel discipline of biotechnology dedicated to stem cell therapy for a variety of human diseases will emerge for the benefit of patients’ health. Concerning these issues, our society will have to decide on setting up the proper scientific, legal and ethical guidelines for possible applications in medicine.
Cloning via embryo splitting (twinning)
Mammalian embryo splitting for the creation of
genomically identical twins or multiples has advanced to a variety of applications in veterinary and human medicine (Figure 3). Embryo splitting to create monozygotic twins and multiples has been successfully reported for several mammalian (47,48). Over the past 25 years, further progress in
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Figure 3. Splitting of early embryos (twinning) for various applications in veterinary and human reproductive medicine refining techniques for embryo splitting has improved the success rates for split embryo survival and live-born offspring. In cattle, Canadian researchers reported that from two embryos split at the 4-cell stage, four identical calves were delivered at term pregnancy (49). Also in cattle, Japanese researchers could create monozygotic calves of different ages after time- separated transfer of frozen-thawed twin embryos (50). It has been argued that pregnancy rates and the chance of conception can be increased by transferring twin embryos derived from embryo splitting (51).
In nonhuman primates, embryo splitting at the 8-cell stage has given low twinning results in rhesus monkey (52). Embryo twinning in rhesus monkey has also been attempted by blastomere separation at the 2-cell and 4-cell stage and led to
seven chemical pregnancies, including two twin pregnancies (53).
Human embryo splitting has so far been reported only on genetically abnormal embryos derived from IVF cycles (54). However, none of these split embryos developed beyond the 32-cell stage. In a commentary on embryo splitting, the merits of this technology has been recognized for future applications in reproductive medicine (55).
Before considering possible applications of human embryo splitting in ART, we decided to develop and establish safe techniques for embryo splitting in the mouse system (56). We found that there is a high success rate for obtaining twin blastocysts with good morphology from 2-cell and 4-cell split embryos (Figure 4). Along these lines we asked ourselves, can early embryos be split several times and, if so, how often? To our knowledge
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Figure 4. Blastomere biopsy from 2-cell and 4-cell mouse donor embryo and blastomere reinjection into empty zona pellucida recipients. Twin blastocysts are derived from 2-cell and 4-cell embryo splitting.
serial embryo splitting has not previously been reported in any mammalian species. We therefore investigated the in-vitro potential of mouse embryos after one, two or three splittings with respect to blastocyst development (57). First and second splitting of embryos has yielded high efficiency rates for blastocysts when compared with the third splitting which did not provide any
beneficial advantage. Our data clearly document that first and second embryo splitting increases the multiplication of blastocysts and therefore increases the number of embryos available for potential transfer.
Embryo splitting in ART may be applicable and considered for those patients termed as “low responders” with only a few oocytes being usually
Vol. 13, No. 1, 2008 Illmensee Embryo biotechnology in reproductive medicine 7
Figure 5. Blastomere cloning from 4- and 6-cell mouse embryos. Blastocysts are derived from individually biopsied blastomeres after 5 days in-vitro culture. Outgrowth and formation of cellular colonies from blastocysts that originated from single blastomeres
after 8 days in-vitro culture. recovered after hormonal stimulation and available for ART. Embryo splitting should increase the likelihood for obtaining a pregnancy since more embryos could be made available for intrauterine transfer. For couples with few embryos produced during one IVF cycle, embryo splitting may provide additional embryos for subsequent transfers without having to go through another retrieval cycle. In a report on embryo splitting as a modality for infertility treatment from the Ethics Committee of the American Society for Reproductive Medicine (58), it has been stated that “as long as a couple is fully informed of the risk of such an outcome, there would appear to be no major ethical objection to placing two or more embryos with the same genome in the uterus with the hope of producing a pregnancy”. This report also promoted research on embryo splitting for future applications in human ART “since embryo splitting has the potential to improve the efficacy of IVF treatments for infertility, research to investigate the technique is ethically acceptable” (58). Cloning of embryonic cells (blastomeres)
Australian researchers reported on the
successful biopsy of single blastomeres from early
mouse embryos and their culture in-vitro, using extracellular matrix components for enhanced cell proliferation (59). In a study on human embryos, British researchers successfully isolated cleavage- stage blastomeres that formed trophoblast colonies during in vitro culture (60). American researchers demonstrated that individual blastomeres biopsied from mouse 8-cell embryos were able to generate ES cell lines that maintained a diploid karyotype and their differentiation potential when tested in- vitro and in-vivo (61). Such approach does not require the destruction of the embryos since the biopsied mouse embryos used for ES-cell derivation were able to develop further into healthy mice.
In our recent studies on mouse blastomere cloning, we have investigated the developmental and morphogenetic potential of individual blastomeres for blastocyst formation during their clonal culture in-vitro (62). We could document that individual blastomeres from cleavage-stage embryos yielded high rates of blastocysts of good morphological quality. Outgrowth and the formation of cellular colonies could be obtained from these blastocysts originating from single blastomeres (Figure 5). Further investigations on
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single blastomeres concerning their capacities for embryo and ES cell formation and for their differentiation potential are of utmost importance for stem cell research. As our knowledge about complex cellular and genetic mechanisms during mammalian embryo genesis is advancing, embryo biotechnology will continue to contribute with novel applications in medicine.
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