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First published online March 23, 2006
Stem Cells Vol. 24 No. 7 July 2006, pp. 1628 -1637
doi:10.1634/stemcells.2005-0592; www.StemCells.com
© 2006 AlphaMed Press

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TECHNOLOGY DEVELOPMENT: CONCISE REVIEW

Using Therapeutic Cloning to Fight Human Disease: A Conundrum or Reality?

Vanessa J. Halla, Petra Stojkovicb, Miodrag Stojkovicb

aNeuronal Survival Unit, Department of Experimental Medical Science, Wallenberg Neuroscience Centre, Lund University, Lund, Sweden;
bCellular Reprogramming Laboratory, Centro de Investigacion Principe Felipe, Valencia, Spain

Key Words. Clinical stem cell transplantation • Reprogramming • Human embryonic stem cells • Cloning

Correspondence: Vanessa Hall, Ph.D.,Neuronal Survival Unit, Department of Experimental Medical Science, Wallenberg Neuroscience Centre, Department of Physiological Sciences, Lund University, BMC A10, Lund, Sweden. Telephone: +46 (0) 46-222-0526; Fax: +46 (0) 46-222-0531; email: Vanessa.Hall{at}med.lu.se

Received November 26, 2005; accepted for publication March 17, 2006.
First published online in STEM CELLS EXPRESS   March 23, 2006.


   ABSTRACT
Top
 Abstract
Introduction
 Conclusion
 Disclosures
 Acknowledgments
 References
 
The development and transplantation of autologous cells derived from nuclear transfer embryonic stem cell (NT-ESC) lines to treat patients suffering from disease has been termed therapeutic cloning. Human NT is still a developing field, with further research required to improve somatic cell NT and human embryonic stem cell differentiation to deliver safe and effective cell replacement therapies. Furthermore, the implications of transferring mitochondrial heteroplasmic cells, which may harbor aberrant epigenetic gene expression profiles, are of concern. The production of human NT-ESC lines also remains plagued by ethical dilemmas, societal concerns, and controversies. Recently, a number of alternate therapeutic strategies have been proposed to circumvent the moral implications surrounding human nuclear transfer. It will be critical to overcome these biological, legislative, and moral restraints to maximize the potential of this therapeutic strategy and to alleviate human disease.


   INTRODUCTION
Top
 Abstract
 Introduction
Conclusion
 Disclosures
 Acknowledgments
 References
 
Currently, cell replacement therapies using allogeneic human embryonic stem cells (hESCs) have been thwarted by the host immune response, which can only be overcome by administering long-term immunosuppressive drug therapy. However, the generation of patient-specific human nuclear transfer embryonic stem cell (hNT-ESCs) lines is a strategy that may circumvent immunorejection. This autologous approach has been circulating in the scientific and media circles since the late 1990s. However, until recently, it was restricted to discussions relating to ethical concerns and potential benefits [1, 2]. NT is an embryo technology that has been used for the purposes of reproductive cloning and, more recently, for therapeutic cloning [3]. In 2004, the realization that patient-specific hESCs could be produced arose following the report of a patient-specific embryonic stem cell (ESC) line produced from a human NT blastocyst; however, this was recently shown to be fraudulent following external review, and the paper was retracted, along with a later publication claiming the production of 11 patient-specific NT-ESCs. It is regrettable that these papers were fraudulent, as they had brought the concept of therapeutic cloning closer to reality. However, other researchers are actively pursuing the generation of NT-ESCs as an alternate source of cells for cell replacement therapies. The implications of the retracted papers on the field of therapeutic cloning and ESC research at such an early stage are unknown. Continuing negative media and public response could be detrimental to this field of research and lead to financial withdrawals and/or lack of funding opportunities, although it is hoped that more positive opportunities may arise. The development of autologous ESCs, which are genetically identical to patients suffering from currently incurable diseases, may provide alternate clinical treatments. Patient-specific hNT-ESCs could be derived from NT blastocysts, which are produced following fusion of a single donor cell from a patient into an enucleated oocyte supplied by a female donor (Fig. 1). Embryonic stem cells have the capacity to self-renew and remain in a pluripotent cell state but also may differentiate into cells representative of the three embryonic germ layers (endoderm, mesoderm, and ectoderm) in the presence of various physical and biochemically inducing factors. Their characterization, culture, and applicability for cell replacement therapy has been well documented [4–6]. This review highlights the progression and development of NT-ESCs in both animal models and in the human. An emerging controversy over the use of NT blastocysts, which contain a heteroplasmic source of mitochondria and which may also harbor altered genetic profiles as a consequence of incorrect nuclear reprogramming, is also raised. Moreover, the current ethical climate and current policies are discussed and how they limit and direct current as well as future research. In light of the ethical concerns, a number of alternative strategies propose to bioengineer cells without the necessity of sacrificing early stage human embryos. The potential and limitations of these strategies are discussed.


Figure 1
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  		Figure 1. Use of therapeutic cloning to treat patients suffering from disease. A fibroblast cell line is isolated following a skin biopsy obtained from a patient and used to create a genetically identical embryo following nuclear transfer (NT). Oocytes are obtained from consenting females and are enucleated to remove the genetic material. A single fibroblast cell is fused into the resultant cytoplast and allowed to undergo nuclear reprogramming. Successful reprogramming and nuclear remodelling results in further embryonic development. The inner cell mass is isolated from the NT blastocyst to establish an NT-ESC line. Differentiation of NT-ESCs into cells suitable for cell transplantation is performed. Following large-scale harvestation, the differentiated NT-ESCs are injected into the patient at the site of interest. Abbreviation: NT-ESC, nuclear transfer embryonic stem cell.

 
Studying NT-ESCs in Animal Models
The production of NT-ESCs has been successful in the mouse. The first reports of the generation of murine NT-ESC (mNT-ESC) lines was published in 2000 [7, 8], with other, similar publications following suit [9–11]. There have been no reported cases of autologous cell transplantations using NT-ESCs. However, the first syngenic transplantation study using mNT-ESC lines was performed in 2002 [10]. Using an Rag2–/– immunodeficient mouse model, researchers were able to produce an mNT-ESC line, which was genetically rescued for the Rag2 allele by performing homologous recombination. Hematopoietic precursors were differentiated in vitro from the repaired NT-ESC line and transplanted into double mutant Rag2 and interleukin-2 common cytokine receptor {gamma} chain ({gamma}C) mice (mice devoid of natural killer cells), which partially rescued their phenotype. Results were limited, however, due to an engraftment barrier and the preferential differentiation into myeloid tissue. The failure to form mature T-cells indicated only partial restoration of immune function [10]. This paper and others [12, 13] indicate that immune rejection following transplantation of nongenetically identical stem cells remains a serious issue; however, this paper is the first to show that the noninvasive isolation of tail tip cells from mice could provide a suitable autologous source of donor cells [9].

Potential of Differentiated NT-ESCs to Treat Disease
Differentiation of NT-ESCs into specific cell types could help to alleviate disease, although to date, relatively few studies have been performed. Allogeneic ESC transplantation studies, however, provide insight into nonhuman animal models and indicate that ESC replacement therapy is a promising tool for future medicine. One such study investigated whether murine ESC (mESC)-differentiated dopamine (DA) neurons could function in vivo following transplantation into a Parkinson’s mouse model phenotype [11]. The alleviation of behavioral deficits was illustrated up to 8 weeks after cell transplantation through the evaluation of the amphetamine- and apomorphine-induced rotation response in mice engrafted with the in vitro-produced DA neurons [11]. Probably the most pressing cell transplantation study to date, performed in nonhuman primates, investigated the in vivo function of allogeneic 5-bromo-2'-deoxyuridine-labeled neural progenitors obtained from cynomologus monkey ESCs following transplantation into the putamen of the Parkinson’s phenotype monkey model [14]. Assessment of cell transplantation using neurological scores on behavior and immunohistochemical analysis up to 14 weeks post-transplantation confirmed the presence and survival of dopamine neurons and dopamine transporter cells. Improvement in posture and motility was also observed approximately 10 weeks following transplantation and remained until 12 weeks post-transplantation, but not in head-checking scores [14]. Many other studies have investigated the effects of differentiated hESCs [15, 16] and mESCs [17, 18] following transplantation into nonhuman animals; these studies indicate moderate graft survival and functional recovery. These studies should be reproduced using NT-ESCs. In addition, longer trials are required to assess the long-term impact of ESC transplantation and potential alleviation of symptomatic disease. Although some results from these studies are promising, high rates of cell death following transplantation are often observed, which remains a major hurdle for cell transplantation therapies. Whether cell survival could be improved using genetically matched cells such as NT-ESCs warrants further investigation.

Progress in Production of Human and Primate NT-ESC Lines
In May 2005, a valid report of the production of an hNT blastocyst was achieved following fusion of an undifferentiated hESC. Unfortunately, the embryo failed to result in the establishment of an NT-ESC line [19]. In this study, oocytes were recovered from a variety of sources, including 1) oocytes that failed to fertilize following either in vitro fertilization (IVF) or intracytoplasmic sperm injection infertility treatment; 2) superstimulated oocytes recovered following follicle reduction (a procedure used to reduce the number of superstimulated oocytes, prior to egg collection); and 3) immature oocytes harvested during oophorectomy/hysterectomy [19]. From this article, it appears that varying oocyte sources may influence NT outcomes. Other developments to date include the production of early cleavage stage hNT embryos produced from oocytes obtained that failed to fertilize (16–18 hours postinsemination) [20]. To date, no data have supported the use of aged/failed to fertilize oocytes for use as donor oocytes for NT [19, 20]. Reported efficiencies for producing hNT embryos are low (Table 1), which may reflect species-specific differences and quality of oocytes obtained. It is clear that further refinement of hNT is required to derive hESC lines.


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  		Table 1. Human nuclear transfer efficiencies reported by either varying donor cell types or sources of oocytes used

 
Nonhuman primate efficiencies by comparison also yield lower NT blastocyst development rates [21] compared with other species. Attempts at reproductive cloning in the primate have resulted in little success, which is thought to be attributed to misaligned chromosomes and defects in microtubule kinetics in the resultant NT embryos, which may be due to the removal of microtubule proteins such as nuclear mitotic apparatus and kinesin-related protein (HSET) during enucleation [21, 22]. Primate NT offspring have been derived following embryonic NT [23], although no pregnancies have been established using more differentiated cell types [21]. In addition, no NT-ESC lines have been reported from nonhuman primates. The establishment of such lines is necessary to study the effects of transplanted primate undifferentiated and differentiated ESCs and an imminent process if we are to extrapolate experimental results to the human clinical setting.

Limitations in Production and Use of Human NT-ESC Lines
The clinical use of human NT-ESC lines is still not feasible due to a number of limiting factors. These limitations are being addressed by several research groups, and new publications are continuously arising in the literature. Limitations that affect both NT-ESCs and ESCs in general include the inability to 1) produce ESC lines in completely defined, animal-free conditions to circumvent the transfer of xenogeneic pathogens from other species [24]; 2) induce differentiation into distinct cell populations that function in vivo [18, 25–27]; 3) purify and isolate homogenous cell populations; 4) overcome the risks in site-specific teratoma formation; and 5) expand the growth of differentiated hESCs within a short time frame. The clinical development of using hESCs and NT-ESCs relies on safe and reliable techniques of cell transplantation and future research is required to optimize the most effective strategies for transferring stem cells into site-specific regions and to determine how many cells would be required for efficient effects. Limitations pertaining to the production of NT-ESCs include legislative constraints, ethical dilemmas, and lack of access to high numbers of human oocytes from fertile, young women. These issues are discussed in detail below.

Ethical and Societal Concerns
Of concern is the possible commodification of human eggs and the creation of a commercial market that would see a price being placed onto "potential human life." Already in the United States, oocytes can be purchased for a cost of U.S. $1,000–$2,000 from consenting women or from volunteer programs where the woman is reimbursed for the clinical procedures in infertility treatments [28]. If the apparent success of producing NT-ESCs is heavily reliant on the age and fertility status of the patient donating the oocytes, this gratification system could become widespread. To date, in valid reported cases of human NT (hNT), women have not been paid for their supply of oocytes other than toward the costs of the superovulation treatment [19]. Although, if the clinical use of NT-ESCs was to become an applicable clinical treatment, would it be appropriate for women to endorse payment for the harvesting of their finite supply of oocytes? "Egg sharing" occurs in some countries, including India, the U.S., and Canada, as well as the U.K.; it allows women undergoing infertility treatment to donate a number of their oocytes to another infertile woman, in return for subsidized fees for treatment [29]. Recently, however, it has been proposed as a possible means for obtaining oocytes for research [30]. Improving the efficiency of deriving patient-specific stem cell lines from NT blastocysts may reduce the quantity of donated oocytes needed for research and help curb the ethical problems of using large numbers of human eggs for research.

Access to high numbers of good quality human oocyte sources is also a current limitation. Within the U.K., the Human Fertility and Embryo Authority has approved a license that allows consenting women undergoing infertility treatment to donate two of their fresh oocytes for research, if more than 12 oocytes are obtained during egg collection. This steady supply of oocytes is limited by the number of consenting IVF patients; the oocytes obtained are from women with a mean age of 32 (our unpublished data). Current hESC lines may be induced to produce oocytes in vitro, following recent reports of the production of oocyte-like cells following mESC differentiation [31], which may help to overcome the ethical dilemmas in obtaining human oocytes for NT. Research on mESCs has indicated that functioning oogonia can be produced following in vitro differentiation [32]. In this study, Vasa +ve differentiated mESC aggregates were isolated, cultured and found to secrete estradiol and express steroidogenic markers, as well as produce oocyte-like cells. However, clear demonstration of the full capacity of these oocyte-like cells to fertilize and support further embryonic development is needed [33].

The fear that science moves too rapidly to keep in pace with societal and moral thinking is one notion that continually erupts in the literature [34], although one could argue that societal and moral concern is also too slow to adapt to the technological advancements of science. There is also a justifiable fear of reproductive cloning when there is an absence of legislation. This public fear may be alleviated if NT-ESCs can be successfully generated and used to help treat and/or alleviate diseases. Researchers participating directly in hNT should therefore remain conscious of ethical views on hNT and should be cautious in the presentation and pursuit of the research. This is perhaps even more imminent following the fraudulent claims of the production of hNT-ESCs. That unfortunate event exemplifies how external and internal pressures, as well as slowly forthcoming results from the laboratory, can lead even senior scientists to risk their career to appease the public and to produce in-demand medical treatments. The use of surplus embryos for derivation of hESC lines has continued to be debated against by religious and other antiembryo research groups. Creation of NT embryos appears to have heightened the level of intolerance and is abhorred by many groups. The fundamental argument is that the sacrifice of an early embryo (considered a human life) should be proscribed for medical research, applying to all embryos and not only those derived by NT. Some groups, including traditional Christian groups such as the Vatican, believe that life begins at conception, whereas others, such as Islam and Judaism, consider that life arises only after development of the fully formed fetus and are unopposed to IVF treatment (for husband and wife only) [35]. Other groups and individuals hold varying views on this question. It is clear that this will remain a heavily debated argument for many years to come. To overcome the immoral perspectives associated with NT, the term "singularity" was spawned as a means to describe the event of producing a meaningful outcome, such as life (i.e., an embryo), from a nonmeaningful origin (a somatic cell) [34]. Singularity also denotes the idea of asexual reproduction, which is an unnatural occurrence for our species. Furthermore, the notion that NT embryos are "somatomes" [36], which are derived from an organism that already exists, appears to cast negative judgment on the use of NT embryos. It is certain that the majority of scientists, ethicists, and the wider community deplore the notion of reproductive cloning, and to circumvent the possibility of the technique being used for embryo transplantation, a worldwide ban should be instated.

Perspectives on Current Legislation
The European Parliament holds a relatively conservative view toward therapeutic cloning; this view is advisory and not legislative. However, the European Union (EU) withholds funding projects that seek to perform NT. At present, EU countries hold individual policies that are either permissive or nonpermissive for researchers to perform human NT for medical research, although all EU member states have signed the Charter for Fundamental Rights [37], which prohibits reproductive NT. Unlike the European Parliament, the European Commission appears more flexible in its opinion on hNT and recently called for a debate to consider the ethical and medical considerations concerning hNT. The outcomes of this debate may solidify or ratify views on such research. Currently, 4 of the 20 EU member states, including Sweden, Belgium, Finland, and the U.K., have passed legislation to allow researchers to perform human cloning for therapeutic purposes only, although restrictions apply. In addition to this legislation exists the Oviedo Convention (1997) and the Protocol on Cloning (1998), which have been signed by a number of member and nonmember states of the Council of Europe (Table 2). In particular, Article 18 of the Oviedo convention states: "1. Where the law allows research on embryos in vitro, it shall ensure adequate protection of the embryo. 2. The creation of human embryos for research purposes is prohibited." In addition, Article 1 of the Protocol on Cloning states "1. Any intervention seeking to create a human being genetically identical to another human being, whether living or dead, is prohibited." Some countries, such as France and Germany, remain heavily opposed to human somatic cell NT, and these countries have put forward a proposal to the United Nations calling for a worldwide ban to take effect in September 2006. The ambiguity of the Oviedo Convention is apparent since signing member states, such as France and Sweden, have very different legislation governing hNT, and nonsigning countries, such as Germany, remain opposed to any form of hNT.


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  		Table 2. Signatures and ratifications of member and nonmember states within the Council of Europe for the Oviedo Convention (1997) and Protocol on Cloning (1998)

 
The United States turned a corner when federal funds were granted to be spent on ESC research; however, researchers are restricted to stem cell lines that were derived prior to August 9, 2001 [38]. However, the United States remains fixed in its stance on not releasing government funding for projects performing hNT, although no restrictions apply to prevent private funders from supporting such research [39]. The President’s Council on Bioethics was formed in January 2002 to monitor stem cell research and to advise on guidelines and regulations in place [39]. This initiative has not prevented researchers, including those at Harvard University, from embarking on hNT research in the U.S. [40]. Currently, legislation pertaining to the use of ESCs and hNT is determined by individual states.

In Asia (China, South Korea, and Singapore), legislation supports the production of human somatic cell NT embryos for medical research. Although swamped in controversy, the scientific endeavor from South Korea (governed by legislation of the Korean National Assembly and answerable to the Korean Bioethics Association) was funded by an estimated annual sum of U.S.$992,000. Following recent evidence of fraudulent claims of patient-specific stem cell lines from South Korea, it is unknown whether funding for such research in Korea will continue.

The view that lagging laws impede scientific advancement and restrict research that could help to alleviate or cure currently incurable diseases is held with contempt by those opposed to research on embryos, ESCs, or hNT. It is apparent, however, that scientists performing hNT have adapted their research to suit government legislation and licenses, which is more restrictive in some countries, such as the U.K., than in others, such as South Korea. Of greatest concern for legislation relating to hNT is the "slippery slope" of current laws governing hNT. The fear is that if laws are permitted to perform hNT, in some years they may slide toward a permissive state to performing reproductive cloning. Some researchers argue that the current implantation inefficiencies of nonhuman primate NT may reflect the difficulties of hNT embryos to implant or even develop to full term [34]. In addition, the production of Cdx2-deficient murine NT blastocysts has been recently proposed as a strategy that would circumvent the implantation of NT embryos [41].

Mitochondrial Heteroplasmy in NT-ESCs
The development of patient-specific ESC lines is considered an autologous approach that would not elicit an immune response from the host; however, little consideration has been made of the possibility of mitochondrial DNA (mtDNA) heteroplasmy (allogenic oocyte mitochondria and autogenic donor cell mitochondria) within hNT embryos and whether these heteroplasmic mtDNA populations remain in the ESC lines following blastocyst derivation (Fig. 2). There is no study to date that has investigated mitochondrial heteroplasmy in hNT embryos, although research in nonhuman animal species has demonstrated that mitochondrial heteroplasmy is prevalent in NT embryos and offspring [42–44]. Interestingly, heteroplasmy has not been observed in all NT offspring [45–47] and has not been observed in NT sheep [48]. Surprisingly, the NT sheep contained mitochondria from the donor oocytes and no contribution from the donor cell; thus, although a homoplasmic source of mitochondria was determined, it was not from the donor cell source [48]. Whether species-specific differences exist that alter the uptake or actively destroys mitochondria from the donor cell following cell fusion, or whether technical variations of NT may be at work, needs to be investigated further. Thus, the medical implications of heteroplasmy remain an issue to consider in the case of hNT [49].


Figure 2
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  		Figure 2. Mitochondrial heteroplasmy in human somatic cell nuclear transfer (NT) embryos. Cell division following NT leads to the unequal distribution of mitochondria derived from the oocyte (pink) and the donor cell (blue), resulting in mitochondrial heteroplasmy in the resultant embryo, which is used for the production of NT embryonic stem cells. Abbreviation: ICM, inner cell mass.

 
Mitochondrial Heteroplasmy and Possible Role in Immunorejection
It should be considered that allogeneic mitochondria present in NT-ESC or NT-ESC derived cells could be recognized by the host immune system, leading to disrupted mitochondrial membrane potential that induces the apoptotic cell signaling pathway [50–52], thus leading to cell death. Although there have been no direct studies to investigate this, one study in the bovine has evaluated whether transplanted NT fetal cells (genetically identical to the original donor) could elicit a T-cell response specific for mtDNA-encoded minor histocompatibility antigens (miHAs) [50]. A maximum of two miHA peptides were identified within grafted recipients, although no T-cell response was determined in response to transplanted cardiac, skeletal tissues or NT derived renal cells [50]. The mitochondrial genome is known to encode a number of transplantation antigens that could trigger an immune response from the host tissue following engraftment [53, 54], such as the maternally transmitted Mta model minor histocompatible antigen [55]. This hypothesis has also been considered following publication of a research paper that linked failed cardiomyocyte grafts with mitochondrial-induced cell apoptosis in rats [51]. The signaling events that induce apoptosis may be linked to the mitochondrial permeability transition (mPT) [51, 52], although other proposed mechanisms have also been elucidated [56]. The mPT occurs via an overload of Ca2+ ions, which induces the opening of a large pore within the mitochondrial inner membrane, which in turn results in a loss of transmbembrane potential ({Delta}{Psi}m) and elicits a signal cascading effect that results in DNA fragmentation and cell apoptosis [57]. Therefore, the possibility that transplanted NT-ESC-derived cells may undergo apoptosis following an immunoresponse cannot be excluded (Fig. 3). This potential immunorejection response needs to be carefully evaluated. It has been proposed that the Ca2+ influx could be prevented using the immunosuppressor cyclosporine A [58], although the advantage in using autologous NT-ESCs for cell transplantation is negated, with the exclusion of immune-privileged sites such as the brain, eye, and testis [59].


Figure 3
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  		Figure 3. Possible mechanism of acute graft rejection due to mitochondrial heteroplasmy following cell transplantation. Loss of {Delta}{Psi}m is induced following the influx of Ca2+ ions into mitochondria, which opens a large pore in the inner membrane and initiates the release of apoptotic factors such as smac, cytochrome c, and AIF. This results in nuclear DNA fragmentation and cell apopotosis. This signaling may occur due to mitochondrial heteroplasmy in transplanted, differentiated NT-ESCs and ultimately result in acute graft rejection. Abbreviations: AIF, apoptosis-inducing factor; smac, second mitochondrion-derived activator of caspase.

 
Possible Transmission of Host Cell Mitochondrial Mutations
Mitochondrial DNA is also known to carry a number of pathogenic mutations and rearrangements that are transmissible. Thus, there remains the potential for the transmission of mutant mtDNAs from the host ooplasm, which could lead to mitochondrial dysfunction and possible disease. Currently, more than 12 clinical disorders are attributed to mutations in the mitochondrial genome [60], although more than 150 point mutations and innumerable large scale rearrangements have been characterized [61]. The pathophysiology of many mitochondrial related diseases also remains largely unknown [61]. Although the transfer of mitochondrial DNA from the paternal genome can occur during natural fertilization, this event is rare, with paternal mitochondria actively being destroyed shortly after fertilization [62]. Reproductive technologies such as embryo aggregation, pronuclear transfer, and NT mediate a higher potential for creation of heteroplasmic sources of mitochondria in the resultant embryo. Cell transplantation studies using donor cells from NT offspring into nonhuman animals may provide useful research into the effects of mtDNA heteroplasmy and the potential immunoresponse and/or risks associated in transfer of mitochondrial related diseases. Alternately, prescreening of female oocyte donors for mitochondrial mutations may need to be considered.

Genetic and Epigenetic Effects
Also of concern are the effects of incorrect epigenetic and nuclear reprogramming of the somatic genome following NT. Epigenetic reprogramming occurs within primordial germ cells and during fertilization [63], but also during NT. Aberrant gene expression patterns have been detected in a high proportion of nonhuman NT embryos compared with IVF-produced and in vivo-produced embryos [64–68]. In addition, inefficient demethylation patterns and incorrect establishment of DNA methylation (precocious de novo methylation) have been detected in NT embryos from many nonhuman animals [63, 69–71]. Other epigenetic defects observed in NT embryos include altered patterns of X inactivation [72, 73], imprinting defects [74], and altered levels of histone acetylation [71]. It is thought these aberrations lead to the failed implantation and developmental abnormalities that are observed in mouse and bovine NT fetuses and/or live offspring. Altered gene expression in NT embryos has been linked to impaired development of the placenta [68], although it is likely that these specific genetic aberrations would have little effect on the production of NT-ESCs, since they are derived from preimplantation-stage blastocysts. Nevertheless, the production of early stage NT embryos derived from failed to fertilize oocytes has revealed that most are karyotypically abnormal, which results in developmental arrest [20]. Analysis of the genetic expression profiles of NT-ESCs compared with ESC lines derived from IVF embryos may provide some useful evidence as to whether significant differences exist, although altered gene expression patterns may already pose a risk in existing hESCs derived from IVF embryos [75]. The effects of epigenetic defects on NT-ESC differentiation are unclear. Epigenetic profiles of current NT-ESCs should be investigated, and careful evaluation should be made of their capability of differentiating compared with hESCs. To date, differentiation of mouse NT-ESCs has not been reported as impaired, compared with standard ESC lines, although caution of the role that epigenetic defects may play in development pathways should be observed.

Epigenetic defects may also result in altered telomere lengths in reproductive NT offspring [76]. Whether altered telomere lengths within NT-ESCs will affect ESC proliferation is unknown, but unlikely. A report that reproductive NT offspring had shorter telomeres than age-matched controls was first raised in 1999 [77], although this has been disputed following more extensive studies on tissues from NT bovine and porcine offspring [78, 79], and no links between telomere length and adverse effects on animal survival have been shown. In contrast, ESCs express high levels of telomerase and maintain their telomere lengths in vitro [80], allowing them to self-renew and avoid senescence. Thus, it is unlikely that epigenetic alterations will affect telomere length at this stage of development. Although no direct studies have investigated telomere lengths of nonhuman NT-ESCs, their ability to self-renew in vitro, like their ESC counterparts, suggests that telomere lengths are maintained. The possible effects of altered epigenetic reprogramming on telomere length in NT-ESCs may only be studied upon directed differentiation and cell transplantation.

Alternative Cell Technologies for Cell Therapy

Adult Stem Cells.   An alternate autologous system to treat various diseases is the harvesting of a patient’s own stem cells (adult stem cells) from sources such as the bone marrow, which may be purified in vitro into cell populations for cell differentiation and later cell transplantation. In contrast to pluripotent ESCs, adult stem cells (ASCs) are multipotent cells (epiblast-like, germ precursor, and progenitor cells) that can differentiate into only a finite number of cell types [81, 82]. However, the ability to improve directed in vitro differentiation is still being pursued by researchers. The use of ASCs certainly circumvents the issue of immunorejection, and ASCs are already being used an alternate cell replacement therapy for bone marrow transplantation and ischemia. However, recently it was discovered that ASCs may spontaneously transform following long-term in vitro culture [83], which warrants further investigation. The literature on ASCs is diverse and can be reviewed more extensively in recent publications [82, 84, 85].

Overcoming Immunorejection Following Transplantation of Allogeneic mESCs.   It has been proposed that immunorejection could be circumvented in non-patient-specific stem cell lines by replacement of the major histocompatibility complex genes [86] with host-specific genes via homologous recombination technology [87]. Immunosuppressive strategies such as overexpression of Fas-ligand in ESCs [88] or knocking out B7 antigens such as CD40 may also overcome the inability to use noncompatible stem cell lines, although these remain a complex molecular challenge and raise issues which are not as straightforward in overcoming as originally proposed, as recent evidence suggests that immune rejection endures following suppression of the CD40 pathway [89]. Furthermore, recent proposals to generate histocompatible ESC banks, comprised of a limited number of ESC lines, may be an effective alternate strategy [90]; however, developing stem cell banks comprised of histocompatible NT-ESCs may be even more advantageous.

Reprogramming Patient Cells by Cell Fusion to Create Patient-Specific Stem Cell Lines.   Recently, it was reported that hESCs could efficiently reprogram human adult skin cells following cell fusion and the formation of stable heterokarons [91]. This technology may serve to overcome the use of human oocytes to reprogram adult skin cells in the future for production of hESC lines. Research into cell fusion and heterokaryon formation has been used to study nuclear reprogramming events and gene expression [92, 93]. Heterokaryon formation may also be a naturally occurring event, as fused cell types have been observed following transplantation of mouse bone marrow-derived cells into adult mouse brains [94] and liver [95, 96]. Although this technology remains hampered by the inability to efficiently remove the original embryonic stem cell nuclei from the heterokaryon cell in vitro and with fusion such a relatively rare event, it seems difficult to envisage the efficiencies in producing large number of reprogrammed cells of normal diploid karyotype.

Use of Parthenogenetic Embryos to Create Female Patient-Specific Stem Cell Lines.   The use of parthenogenetically activated embryos for the creation of female haploid ESC lines could serve as an autologous source of cells for producing differentiated cell types to treat women suffering from diseases such as Type 1 diabetes or spinal cord injury [97]. In addition, the creation of these haploid blastocysts from the patient’s own oocytes sidesteps various ethical dilemmas such as sacrificing normal embryos (capable of implanting and developing further) or the creation of NT embryos. The production of a parthenote nonhuman primate ESC line demonstrates the feasibility of this strategy [98], but although the cell line remained karyotypically normal and differentiated into all three germ layer cell types in vitro and in vivo, the functioning of the haploid differentiated cell types in vivo remains unknown and needs to be extensively characterized. In the human, a near haploid cancer cell line has been cultured and remained karyotypically stable for 8 months [99].


   CONCLUSION
Top
 Abstract
 Introduction
 Conclusion
Disclosures
 Acknowledgments
 References
 
In summary, the production of patient-specific stem cell lines is closer to reality, which will help to provide an alternative source of hESCs for treatment of disease. Although extensive research is required to streamline hESC differentiation, improve hESC cultures, and improve cell transplantation methodologies. The recent advancements in hNT and animal cell transplantation using NT-ESC lines may help to bring this technology to a clinical platform sooner than we might think. In addition, further research into mitochondrial penetrance from donor oocyte sources and possible effects that aberrant gene expression patterns may have within hNT blastocyst and NT-ESC lines is required. An increase in the number of countries that permit human NT may allow faster progress in improving efficiencies, although many countries already allow such research to occur, and this is likely to remain a changing landscape.


   DISCLOSURES
Top
 Abstract
 Introduction
 Conclusion
 Disclosures
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Conclusion
 Disclosures
 Acknowledgments
References
 
We thank Simon Foster for the compilation of the illustrations in this manuscript.


   REFERENCES
Top
 Abstract
 Introduction
 Conclusion
 Disclosures
 Acknowledgments
 References
 

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