Human Therapeutic Cloning (NTSC) - Stemagen French et al.pdf · 2013-02-21 · Human Therapeutic Cloning (NTSC) _____267 Stem Cell Reviews ♦ Volume 2, 2006 The reasons for the apparent

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Human Therapeutic Cloning (NTSC)Applying Research From Mammalian Reproductive CloningAndrew J. French,*,1 Samuel H.Wood,1,2 and Alan O.Trounson3

1Stemagen Corporation, La Jolla, California, USA 92037; 2The Reproductive Sciences Center, La Jolla California,USA 92037; and 3Monash Immunology and Stem Cell Laboratories, Monash University,Victoria 3800 Australia

AbstractHuman therapeutic cloning or nuclear transfer stem cells (NTSC) to produce patient-specific stem cells, holds considerable promise in the field of regenerative medicine. Therecent withdrawal of the only scientific publications claiming the successful generationof NTSC lines afford an opportunity to review the available research in mammalian repro-ductive somatic cell nuclear transfer (SCNT) with the goal of progressing human NTSC.The process of SCNT is prone to epigenetic abnormalities that contribute to very lowsuccess rates. Although there are high mortality rates in some species of cloned animals,most surviving clones have been shown to have normal phenotypic and physiologicalcharacteristics and to produce healthy offspring. This technology has been applied to anincreasing number of mammals for utility in research, agriculture, conservation, and bio-medicine. In contrast, attempts at SCNT to produce human embryonic stem cells (hESCs)have been disappointing. Only one group has published reliable evidence of success inderiving a cloned human blastocyst, using an undifferentiated hESC donor cell, and itfailed to develop into a hESC line. When optimal conditions are present, it appears thatin vitro development of cloned and parthenogenetic embryos, both of which may be uti-lized to produce hESCs, may be similar to in vitro fertilized embryos. The derivation ofESC lines from cloned embryos is substantially more efficient than the production ofviable offspring. This review summarizes developments in mammalian reproductivecloning, cell-to-cell fusion alternatives, and strategies for oocyte procurement that mayprovide important clues facilitating progress in human therapeutic cloning leading tothe successful application of cell-based therapies utilizing autologous hESC lines.

Index Entries: Cloning; embryonic stem cells; human; oocyte donation; somatic cellnuclear transfer; therapeutic cloning.

Stem Cell ReviewsCopyright © 2006 Humana Press Inc. All rights of any nature whatsoever are reserved.ISSN 1535–1084/06/2:265–276/$30.00 (Online) 1558–6804

amid allegations of scientific impropriety,of publications that claimed successfulhuman therapeutic cloning (1), it wouldseem an opportune time to regroup and toreview the available data from mammalianreproductive SCNT research that may ulti-mately suggest new strategies to progressthe goal of therapeutic cloning in humans.As a starting point, it is useful to re-examinethe parallels and recent developments in

IntroductionHuman “therapeutic cloning” describes

the use of somatic cell nuclear transfer(SCNT) to produce patient-specific stemcells. If scientific advances allow therapeu-tic cloning to be performed in a consistentand efficient manner, its potential in under-standing and developing treatments fordegenerative diseases is potentially limit-less. Given the recent voluntary withdrawal,

*Correspondence and reprintrequests to:

Andrew French,Stemagen Corporation,La Jolla, CA 92037.E-mail: [email protected]

*According to the International Society for Stem Cell Research’s (ISSCR) NomenclatureStatement (September 2, 2004).

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mammalian reproductive cloning that may ultimately providethe conduit to the promise of safe and effective patient-specificpluripotent cells for cell therapies, and tissues for regenera-tive medicine (2–8). Human nuclear transfer (NT) remains inits infancy, with the one reliable report demonstrating the gen-eration of a single cloned blastocyst from an undifferentiatedhuman embryonic stem cell (hESC) donor cell, and it failed toreinitiate into a hESC line (9).

This review highlights recent strategies in mammalian biol-ogy that improve the efficiency of nuclear transfer and nuclearreprogramming. These strategies include the use of pluripotentcells as donor cells and the treatment of donor cells with cellu-lar extracts to increase the access of reprogramming factors todonor chromatin. Potential approaches to the challenging prob-lem of procuring high-quality oocytes for use in the generationof cloned human embryos is the critical first step toward theaccomplishment of SCNT for patient-specific ES cells.

Reproductive CloningThe restoration of totipotency to a differentiated nucleus

following fusion with an enucleated oocyte, to produce healthycloned animals, has been progressively applied to an increas-ing number of animal species using a variety of donor celltypes (10–13). Although remarkable, the process is highly inef-ficient and subject to epigenetic errors that result in high ratesof morbidity and mortality throughout development.Although the potential applications of SCNT in research,industry, and animal breeding are vast (10,14–19), they remainhampered by the requirement to achieve competitive com-mercial outcomes. To date, SCNT, in combination with trans-genic strategies, has been applied to laboratory and domesticanimals in the areas of:

1. Basic research to generate animal models of human dis-ease to further our understanding of biological eventssurrounding development and functional genomics;

2. Animal breeding programs to increase biological effi-ciency at a reduced cost;

3. Preclinical testing of new therapeutic interventions forhuman medicine; and

4. Genetic rescue of endangered mammals by cross-speciesSCNT.

To achieve full-term development, the cloned embryo hasto overcome a significant number of molecular and cellularchallenges, including the regulatory environment of the recip-ient oocyte, the plasticity of reprogramming in the donor cell,recipient and donor cell cycle coordination, technical compe-tence of micromanipulation, chromatin remodeling and repro-gramming of gene expression, parthenogenetic activation ofthe reconstructed cell, and exposure to culture conditions usedto reinitiate embryonic development.

The rate of generation of cloned offspring by nuclear trans-fer remains low in all species, irrespective of donor cell type(20–23). In many species, and within strains of species, it remainstechnically challenging to produce cloned offspring.Conversely, nuclear transfer in the mouse and bovine has beenused successfully to generate ESC lines from somatic cells withrelative ease using a variety of genotypes and cell types fromboth genders (24,25). These ESCs have been used to rescue

immune-deficient phenotypes (26); however, it remains to bedetermined if ESC lines have the same functional characteris-tics as those derived from fertilized embryos. Recent evidencein the mouse suggests a high degree of equivalency betweennuclear transfer stem cell (NTSC)-derived ESCs and those fromfertilized mouse blastocysts (27). Collectively, the data indi-cates that the generation of ESCs from NTSC (therapeuticcloning) is far more efficient (up to 10 times higher in the mouse)and hence less exacting, than the demands for complete devel-opment to term of SCNT reconstructed embryos (28–32).

Mammalian SCNTAlthough significant variations exist, the basic features of

SCNT in both laboratory and domestic animals involve the trans-plantation of a diploid nucleus into a mature oocyte cytoplast.The procedure was first successfully accomplished in mammalsin 1996 with the birth of a cloned lamb (33) that established thatcell differentiation and commitment to end-stage tissue typecan be reversed to enable complete resetting of the embryonicdevelopmental program. To date, reproductive cloning has beensuccessfully used to derive offspring in 12 species using approx12 different donor cell types from among the approx 210 adultdifferentiated cell types that exist in mammals (22). Some celltypes, including fetal fibroblasts (11,34), ESCs (35–37), andoviduct epithelial cells (38), appear more suitable as donor nucleifor generating offspring than others, although this hypothesishas not been conclusively established (39,40).

Around 6% of all embryos transferred to the synchronizedreproductive tracts of surrogate mothers result in healthylong-term surviving clones (41). The majority of clonedembryos fail to thrive as a result of developmental anomaliesin both the fetus and placenta (see Fig. 1). These developmen-tal abnormalities are likely to be as a result of aberrations ingene regulation mechanisms, including errors of epigeneticreprogramming of the donor genome following nuclear trans-fer (35,36,42–44) and are later manifested as precocious orabsent patterns of gene expression during development.Abnormalities have been detected in DNAmethylation (44–46),chromatin modification (47), X-chromosome inactivation(48,49), and expression of imprinted and nonimprinted genes(50–56). Even apparently healthy cloned animals show epi-genetic defects affecting the expression of genes activated laterin development or adulthood (57).

A likely source of variability may be species-specific,including but not limited to speed of development, responseto parthenogenetic activation stimuli, and differing meta-bolic requirements during in vitro culture, or they mayderive from variations in the methodologies used. The prog-eny from clones appear to be free of both developmentaland phenotypic anomalies (41,54). The physiology of sur-viving postpubertal cloned bulls and the quality of theircollected semen apparently share equivalent reproductivepotential to their original cell donor with no evidence of anydeleterious effects in their progeny (58–60). Although con-firmation is required at the molecular level, it appears epi-genetic errors prevalent during in vivo development ofcloned animals are subsequently erased during gameto-genesis, preventing transmission of aberrant cloning phe-notypes to the offspring of clones.

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The reasons for the apparent markedly reduced develop-mental competence of SCNT embryos when compared with invivo embryos and those from other assisted reproductive tech-nologies such as in vitro fertilization (IVF), has been the sub-ject of considerable scientific debate. Perhaps unexpectedlythen, a direct comparison of the in vitro development rates ofcloned vs IVF embryos may serve to clarify this matter (25,61).Although variation between laboratories and subtle differencesbetween species impact the overall success of reproductivecloning, it appears that once optimalconditions have been estab-lished in any given laboratory, the in vitro development of bothcloned and parthenogenetic embryos approaches that of in vitrofertilized embryos (see Fig. 2; 25,61) and both may be used toderive ESCs. Thus, it may be that attaining the “Gold Standard”for in vitro fertilized embryos will emerge as the key indicatorof proficiency in nuclear transfer programs.

SCNT MethodologiesSCNT is a technically demanding, multistep procedure with

the potential for cumulative errors that can impact develop-mental potential and each stage of development. Althoughnumerous variations exists, the basic features of the SCNT pro-cedure, used in both laboratory and domestic animals, haveremained the same since nuclear transfer in mammals was firstperformed in the early 1980s and can be summarized as fol-lows:

1. Enucleation of the oocyte to form a cytoplast; 2. Merger of the donor cell or nucleus with the cytoplast to

form a reconstructed one-cell embryo; 3. Parthenogenetic activation either before of after recon-

struction of the reconstructed one-cell embryo; and 4. Preimplantation embryo in vitro culture.

Maternal chromosomes are most commonly removed (enu-cleated) by aspirating or extruding a membrane-bound por-tion of cytoplasm containing the metaphase plate, or bybisection containing the metaphase plate. Other methods ofenucleation include centrifugation, inactivation, or destruc-tion by ultraviolet or laser irradiation, or noninvasivelyexpelled by chemically induced extrusion (11). Successful enu-cleation is confirmed by incubating the oocyte in Hoechst33342, a nontoxic DNA interchalating agent, which fluorescesin the presence of ultraviolet light (11). Reconstructed one-cell embryos are formed when the donor cell or karyoplast isdeposited into the perivitelline space and adjacent membranesare fused by exposure to a series of electrical pulses, inacti-vated Sendai virus, or treatment with chemicals such as poly-ethylene glycol. Alternatively, donor nuclei have been directlyinjected into the cytoplasm of the cytoplast in manner akin tointracytoplasmic sperm injection (ICSI).

Oocyte Source and AvailabilityThe rapid progress seen in optimizing the reproductive

cloning of domestic animals was in no small part the resultof the availability of an alternative and almost unlimited sup-ply of developmentally competent oocytes; those harvestedfrom ovaries opportunistically scavenged from abattoir pro-cessing. There is little doubt that the quality of the oocyte,i.e., its intrinsic developmental potential is a key factor deter-mining the proportion of in vitro fertilized or nuclear trans-fer embryos that grow to the blastocyst stage (62), and it alsoinfluences the overall success of any assisted reproductivetechnology. An eloquent demonstration of this effect wasshown when the superovulation protocol in the bovine wasmanipulated to produce an almost maximal number of competent

Fig. 1. Developmental competence of in vivo, IVM/IVF, and IVM/SCNT bovine embryo production systems.

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cumulus–oocyte complexes (COCs) as determined by blas-tocyst formation and significantly improved in vitro embryoproduction systems (63).

SCNT ImprovementsCell Synchronization

Coordination of the cell cycle between the donor cell nucleus(karyoplast) and the recipient cell cytoplasm (cytoplast) isessential to minimize DNA damage and to maintain euploidyin the reconstructed cloned embryo (64). Cloned offspring havebeen obtained using cytoplasts at telophase (second meioticdivision) and interphase of the first cell cycle and at metaphaseII (second meiotic division) (64,65). The readily availablemetaphase II cytoplasts have typically become the recipient ofchoice for SCNT experiments.

Cell cycle inhibitors have been used to increase cell cyclesynchronization. Recent work in the bovine has shown that donor cells treated with a new metaphase arrestor (2-methoxyestradiol) permitted increased recovery of mitoticcells following shaking. When these cells, arrested at early G1,were subsequently used for nuclear transfer, the resulting SCNTembryos were shown to have a higher developmental com-petence (blastocysts and live birth rates) (66).

Chromatin TransferThe general consensus of laboratories performing repro-

ductive cloning in animals is that future improvements aremost likely to come from a greater understanding of the molec-ular mechanisms of reprogramming. In the bovine, a novelsystem allowing remodeling of mammalian somatic nucleiin vitro before SCNT has been developed (67). Donor cellsare permeabilized to allow a mitotic extract access to the chro-matin and to facilitate removal of nuclear factors solubilizedduring chromosome condensation. The condensed chromo-somes are transferred into enucleated oocytes before activa-tion. When compared with nuclear transfer embryos, thesechromatin transfer embryos appear to exhibit a pattern ofmarkers that more closely resembles those of normal IVFembryos.

Initial results indicate that calves born after chromatin trans-fer may have a greater survival (see Fig. 3; A French unpub-lished observations). More importantly, this technique providesa method for directly manipulating the somatic donor chro-matin before transplantation, and thus it may be a useful toolto investigate mechanisms of nuclear reprogramming and toenable improvements to the efficiency of both reproductiveand therapeutic cloning.

Fig. 2. SCNT proficiency in the bovine—a retrospective profile of in vitro production systems to the blastocyst stage. A yearly profile of in vitroproduced bovine blastocysts showing percents of in vitro matured (presence of polar body [PB], metaphase II), in vitro fertilized (IVF blastocystrate), parthenogenetically activated (PA blastocyst rate), and cloned (SCNT blastocyst rate). Linear trend lines shown for each of the four groups(ivm – solid; parthenogenetic – solid; IVF – dotted; SCNT – dashed lines). Modified from ref. 61.




Fig. 3.The efficiency of a generation of bovine SCNT calves using traditional nuclear transfer, chromatin transfer, and hand-made cloning methodolo-gies.The developmental competence of bovine SCNT embryos from a number of somatic fibroblast cell lines using traditional nuclear transfer, hand-made cloning,and chromatin transfer methodologies against IVF embryos from the same laboratory (2003–2005; A French unpublished observations).

Therapeutic CloningThere is a clear distinction between reproductive cloning

and the derivation of embryonic cells for their potential use inregenerative medicine. Human reproductive cloning is notonly unethical, it is illegal throughout much of the world.Therapeutic cloning, on the other hand, is becoming increas-ingly accepted with substantial governmental fundingavailable in some localities. These promising initiatives havebeen guided by principles (68,69) that address the associatedwide-ranging ethical and societal concerns (70–73), that mayultimately permit the accrual of benefits to society thoughimprovements in regenerative medicine. Undoubtedly,continued research designed to improve the efficiency ofreproductive cloning in laboratory and domestic animals,and to develop alternate somatic cell reprogrammingtechniques, will prove useful in parallel studies of method-ologies to increase the efficiency of patient-specific stem cellstrategies.

Therapeutic Cloning in Animal ModelsIn 2000, the utility of therapeutic cloning was first demon-

strated in the mouse, when autologous ESC were derivedfrom a cloned mouse blastocyst (74,75); this observationwas subsequently reaffirmed in a number of similar pub-lications (26,28,76). However, although autologous celltransplantations using isogenic ESCs have not beenreported, synergic transplantations have elicited a partialrescue of a deficient phenotype (26). Homologous recom-bination in the Rag 2–/– allele was used to correct the geneticdefect in a nuclear transfer embryonic-stem cell line derivedfrom this immuno-deficient mouse. Partial rescue of the

phenotype was achieved following transplantation ofhematopoietic precursors differentiated from the correctednuclear transfer ESC line.

Therapeutic Cloning in Human and Nonhuman Primates

At present, only one reliable published report describingthe production of a human NTSC blastocyst exists. Thisblastocyst was derived following fusion of an undifferenti-ated ESC (9) with an oocyte obtained from a follicular-reduc-tion procedure. Unfortunately, this blastocyst failed todevelop into an ESC line. Importantly, this report suggestedthat the oocyte source and age may influence NTSCoutcomes. Other preliminary reports have generated onlyearly cleavage stage nuclear transfer embryos (77b) andusing cytoplasts obtained from aged human IVF oocytesthat had failed to fertilize (77a). Lu et al. (2003) have alsoreported to have cloned human embryos using fetal fibro-blasts and granulosa cells, although the reliability of thefinding is uncertain since no DNA fingerprinting or othergenetic confirmation demonstrating that they were indeedclones was provided (77c).

The generation of SCNT blastocysts in nonhuman primatesappears to be far more inefficient as compared with otherspecies, and attempts at therapeutic cloning have been entirelyunsuccessful. The failures to date have been attributed to mis-aligned chromosomes and defects in microtubule kineticsresulting from removal/interruption of microtubule proteins(nuclear mitotic apparatus protein [NuMA] and kinesin-related protein [HSET]) at the time of enucleation (78,79).However, primate embryonic NT has been used successfully

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to derive offspring (80). The establishment of therapeuticcloning in nonhuman primates could be important for inves-tigating the effects of undifferentiated and differentiated ESCsin transplantation studies and may allow extrapolation tohuman therapeutic intervention. Potential strategies for thederivation of human ESC lines for cell-based therapies areshown in Fig. 4.

Limitations to Human Therapeutic Cloning

SOURCE OF HUMAN OOCYTES

Undoubtedly, a significant impediment to progress in the areaof human therapeutic cloning is the very limited availability ofhigh-quality human oocytes for this purpose. In the human,the emergence of IVF as a highly successful treatment optionfor infertility has given rise to 25 yr of experience in ovarianstimulation (81). The ability in the human to induce multipledominant follicles by gonadotrophin stimulation and to growand produce mature, developmentally competent oocytes hasimproved the chance of conception both in vivo (empiricalovarian stimulation with or without intrauterine insemina-tion) and with IVF (82). Ovarian stimulation permits theretrieval of numerous COCs and thus compensates for

inefficiencies in oocyte maturation, fertilization, in vitro cul-ture, and subsequent in vivo development that might resultfrom having these events occur in vitro rather than in vivo (81).

The success of SCNT will very likely be influenced by theage and fertility status of the woman donating her oocytes, withyounger women without a history of infertility presumablybeing the best donor candidates. Although ethical and legalconsiderations strongly impact the availability of donor oocytes,several prospects exist for consenting patients to provideoocytes for compelling research proposals. To date, sources ofdonated oocytes for research have ranged from altruistic oocytedonation in which no cost or benefit accrues to the donor (9) toa more traditional oocyte-donation model in which donorsreceive compensation, either monetary or in the form of reducedtreatment fees. Although compensation in excess of expensesis controversial and prohibited by law in some localities, a levelof compensation that is not viewed as an undue inducementmight prove to be practically necessary to obtain the quantityof oocytes necessary for research in this area. As has been pre-scribed for reproductive oocyte donation, this compensation,if allowed, would acknowledge the very real fact that ovarianstimulation and oocyte retrieval are not without risk or dis-comfort (83,84).

Fig. 4. ESCs for cell-based therapies.



Source of donated of oocytes for the purposes of SCNT mayinclude the following categories:

1. Oocytes that fail to fertilize following either IVF or ICSI.However, no data exists at present to support evidencethat aged oocytes, or those that have failed to fertilizein IVF or ICSI, provide a suitable ooplasm for repro-gramming donor nuclei (9,77), their utility may berestricted to proficiency testing.

2. Oocytes recovered following follicle reduction, a proce-dure performed to minimize the risk of multifetal preg-nancy following unintended excessive gonadotrophicstimulation during infertility treatment.

3. Immature oocytes obtained from ovarian tissue follow-ing oophorectomy, hysterectomy, and caesarian section.

4. Oocytes obtained after ovarian stimulation and oocyteretrieval that are excess to reproductive requirements (9).

There is a need in some of these categories for effective invitro maturation protocols to be optimized for human oocytes,so that immature oocytes can complete their normal matura-tion at a practical rate, thus allowing fertilization and earlypreimplantation embryonic development to occur. Immatureoocytes recovered opportunistically at surgery and those recov-ered as a proportion of COCs following ovarian stimulation forIVF, also require in vitro culture for cytoplasmic maturation tobe completed. Immature human oocytes obtained from patientsundergoing gynecological surgery or ovulation induction canbe matured and fertilized in vitro (82). Following transfer ofthese embryos to the patient’s uterus, pregnancies and live birthshave been achieved (85). However, the developmental capacityof in vitro matured oocytes is reduced compared with thoseretrieved after maturation in vivo (86,87). An alternate source,one that involves the nurturing of immature oocytes from fetalovarian tissue (88), is unlikely to have ethical support.

As the technology of human SCNT matures and the appli-cation of vitrification techniques for oocyte cryopreservationis increasingly adopted (89), it is foreseeable that cryobanks ofhuman ova available for donation could be established to pro-vide a readily available source of cytoplasts for NTSC and thera-peutic purposes (90). An alternative supply of human oocytesfor NTSC could come from the differentiation of ESCs into germstem cells and putative ova in vitro. Preliminary studies in themouse have shown that oocyte-like cells can be produced fol-lowing differentiation of mouse ESCs (91,92). It does appearthat functioning oogonia express markers restricted to this celltype (91,92), although confirmation of their ability to fertilizeand produce viable young will be required to fully confirm thispotential as a source of reprogramming cytoplasts (93).

Reprogramming and RemodelingCritical to successful SCNT is the resetting or repro-

gramming of the epigenetic modifications that maintain thedifferentiated state of the somatic genome, a state conservedby DNA methylation and by the covalent modification ofnucleosome histones. For embryonic development to pro-ceed, the oocyte cytoplasm must interact with the transferreddonor nuclei to silence expression of its somatic cell-specific

genes and upregulate embryonic-specific genes in the cor-rect spatio-temporal manner. Although relatively little isknown of the cellular factors responsible for remodeling orreprogramming, studies in both Xenopus (94) and mammals(95) have revealed a role for nucleoplasmin in the removalof histones and for the imitation switch in the removal ofTATA-binding proteins (TBP) from DNA (96). The mecha-nisms governing nuclear reprogramming appear to be widelyconserved with oocyte components from different specieshaving the ability to reprogram gene expression in a varietyof somatic cells from other species (97). The remodeling ofnuclear structures by oocyte components results in the reac-tivation of genes associated with pluripotency in earlyembryos and pluripotential stem cells (98). The failure orincomplete silencing of gene products that relate specificallyto the donor cell types can generate profoundly altered char-acteristics that lead to severe consequences for the develop-ment of cloned embryos (99).

Mitochondrial HeteroplasmyThe unfertilized mammalian oocyte contains approx 100,000

mitochondria. Following fertilization and during early cleav-age development, the small number of paternally derived mito-chondria, transferred with sperm, are eliminated by an as yetunknown mechanism. The activity of ubiquitin, and the lyso-somal apparatus of the egg appear to be involved in the pro-teolytic destruction of bovine-sperm mitochondria inside eggcytoplasm, although the mechanism of ubiquitination of spermmitochondria leading to their destruction appears species-specific (100). By the time the blastocyst stage of developmentis reached, cells contain approx 100 mitochondria (indicatingthere is no replication) of entirely maternal contribution(101,102). Rarely, however, mitochondrial heteroplasmia doesoccur in humans (103) and in other mammals (102,104).

Mitochondrial heteroplasmy has been observed followingSCNT in other mammalian species (105–107), although not inall cloned offspring (108,109). The modified somatic cellnuclear-transfer technique, termed “hand-made cloning,”involves the construction of several fused cytoplasts so thatthe SCNT blastocyst, although genomically identical, couldhave mitochondrial DNAcontributions from two to six oocytes(110). In an experiment conducted in the bovine, there was noevidence of any harmful effect of mitochondrial DNA hetero-plasmy or differences in reproductive efficiency when com-pared with the standard nuclear-transfer procedure using asingle oocyte (110). There is a possibility that species-specificor methodological differences could exist that could lead tovariations in the level of mitochondrial heteroplasmy that mayhave implications for human therapeutic cloning and this war-rants further investigation (111,112). Arecently described ubiq-uitination method may represent a mechanism for theelimination of paternal mitochondria during fertilization insome species (100).

Parthenogenetic ActivationCritical to the reprogramming of the donor nucleus and the

development of NTSC embryos is the resumption of meiosisand restoration of cell cycle progression (113). During normalfertilization, sperm penetration activates the phosphoinositide


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pathway (114,115), thereby triggering Ca(2+) release and eggactivation through a prolonged series of intracellular free cal-cium ion oscillations that continue for several hours. Althoughthe precise signaling pathway initiated by the sperm remainsunknown, recent evidence favors the delivery of a solubleprotein factor by the sperm that increases the production ofinositol 1,4,5-triphosphate, which acts to open Ca(2+) chan-nels in the endoplasmic reticulum thereby releasing Ca(2+)into the cytosol.

A variety of electrical and chemical activation methods thatinfluence maturation promoting factor activity and mitogen-activated protein (MAP) kinase activity have been devised toduplicate or closely mimic the changes in the oocyte cytoplasmthat normally are triggered by the sperm during fertilization(116). However, most treatments cause only a single transientCa(2+) spike, which serves to activate only a fraction of targetoocytes, one that increases with the time after ovulation.Attempts to prolong the Ca(2+) oscillations have been successfulwith less mature oocytes but have been unwieldy (117,118).

A popular and highly effective oocyte activation methodinvolves inducing an elevation in intracellular Ca(2+) with acalcium ionophore while maintaining low levels of matura-tion promoting factor using a protein synthesis inhibitor, forseveral hours after the initial Ca(2+) elevation. Regardless ofthe activation procedure utilized, however, both pregnancyand survival rates after birth of cloned animals remain low.Parthenogenetic activation is therefore likely to be a cocon-tributor to the inefficiency of cloning. New activation proto-cols that more closely mimic fertilization and the physiologicalactions of sperm may yield improvements in viability.

Human oocytes, like those of other mammals, can be artifi-cially activated by stimuli that elevate cytosolic Ca(2+) levels,with both fresh and aged oocytes subsequently undergoingpronuclear formation and early cleavage division (119).However, as other studies have found that calcium-ionophoretreatment alone does not activate human oocytes in a reliablefashion (120,121), the commonly used activation protocols inhuman studies also combine a calcium ionophore along withprotein synthesis or protein kinase inhibitors (122–124).Although these combination treatments have induced pronu-clei formation in activated human oocytes, the success ratesin terms of preimplantation development to the blastocyststage are still very poor when compared with embryo devel-opment after in vivo or IVF.

Cytosolic extracts from sperm can cause Ca(2+) oscillationsin a range of different mammalian oocytes, including humans(114,125,126). In a further refinement of this approach, Saunderset al. (127) isolated a novel protein from sperm that is a specificisoform of phospholipase C (PLC), referred to as PLCζ (zeta)(128),which is responsible for generating Ca(2+) release and induc-ing InsP3 production. Homologs for this protein exist in humansand primates (129). Microinjection of cRNA encoding humanPLCζ induced a prolonged series of calcium oscillations in agedhuman oocytes that closely mimicked the repetitive nature ofthe Ca(2+) stimulus provided by the sperm during human fer-tilization. At low concentrations, injection of PLCζ; inducedparthenogenesis and development to the blastocyst stage (130).The use of sperm extracts and PLCζ to activate human oocytesfor the purposes of generating both cloned and parthenogeneticstem cell lines certainly warrants further investigation.

Parthenogenetically activated oocytes themselves representa possible source of pluripotential stem cell lines that are notembryonic. A nonhuman ESC line was established by Cibelliet al. (131,132) from parthenogenetic activation of monkeyoocytes that showed all the characteristics of traditional humanESCs. Recently, Brevini et al. (133) have reported the produc-tion of human ESCs derived from parthenogenetically acti-vated human oocytes. Oocytes were activated by 5 µMionomycin (5 min) and 2 mM 6-dimethylaminopurine (6-DMAP) (3 h). Two cell lines extensively proliferated asdiploid, undifferentiated cells for more than 25 passages andmaintained the expression of pluripotential markers. Theyshowed differentiation as embryoid bodies in the three pri-mary germ layers (endoderm, mesoderm, and ectoderm) invitro. This data suggests that nonembryonic ESCs can be estab-lished from unfertilized human oocytes and this should be fur-ther explored as a source of new pluripotent stem cells.

Human Embryo CultureCulture environment during the preimplantation period of

development profoundly influences the physiology and via-bility of mammalian embryos. Susceptibility to environmen-tal stimuli decreases as development proceeds. The degree towhich abnormal gene expression and altered imprinting pat-terns can be reduced or averted by using more physiologicaland environmental conditions is presently unknown.

Improvements in the success of the assisted reproductivetechnologies for the treatment of infertility have coincided withthe development of highly complex, biphasic culture mediaand culture conditions, which offer improved in vitro devel-opment, pregnancy rates, and live births (134). A multitude ofcommercially sourced media, manufactured under stringentmanufacturing conditions, are available for this purpose.

However, culture media specifically optimized for the devel-opment of SCNT is not yet available. Previous work indicatesthat NTSC embryos may require different culture requirementsas a result of altered physiology and gene metabolism expres-sion pathways (135). Thus, optimizing such culture conditionsmay be important for understanding the kinetics and mecha-nisms of nuclear reprogramming and to improving the over-all process.

Alternative Strategies for Therapeutic CloningCell Fusion Based

The first demonstration that a pluripotent cell couldreprogram a somatic cell following cell fusion to form a sta-ble heterokaryon was in the mouse (136), and this phenome-non has been used to study nuclear reprogramming and geneexpression (137). In an analogous human study, ESCs haverecently been shown to efficiently reprogram human adult skincells following cell to cell fusion (138). The current limitationremains the removal of the original ES cell nuclei to form adiploid isogenic ESC line, although recent work indicates thatthe use of centrifugation both before and after fusion can accom-plish this task (139,140).Spontaneous heterokaryon formationis known to occur in vivo, as fused cell types have been observedfollowing transplantation of bone-marrow-derived cells intoadult mice brain and liver (141–143). Although this technol-ogy has the potential to replace the use of human oocytes in



the reprogramming process, the low rates of spontaneousfusion, the inefficiency of producing large numbers of repro-grammed diploid cells, and a variety of safety concerns mayultimately limit its use (144).

Interspecies Therapeutic CloningThe scarcity of human oocytes available for this type of

research has led some researchers to generate human ESCs byreprogramming somatic cells with oocytes from another species(145). ESCs generated by this process appear to retain the char-acteristics of those derived from human embryos (145). Theapparent universally conserved ability of oocyte componentsto reprogram gene expression in somatic cells is generatingconsiderable interest leading to its exploration in several species(146–149). The utility of this approach requires further inves-tigation. To overcome the concerns of mitochondrial hetero-plasmy, it has been suggested that the ooplasm, mitochondria,as well as other ultrastructural components be replaced withhuman equivalents in a process similar to cytoplasmic trans-fer to allow a generation of blastocyst made up entirely ofhuman material (8).

Extract-Mediated TransdifferentiationThe elucidation of the mechanisms associated with nuclear

reprogramming could eventually lead to a direct reprogram-ming of human somatic cell nuclei without the use of eggs.Such an approach would ameliorate many of the difficultiesassociated with current nuclear transfer procedures and has-ten the generation of replacement cells for therapeutic pur-poses. The demonstration that adult stem cells have broaderdifferentiation potential than anticipated and that they cancontribute to tissues other than those in which they reside,has led to the development of a novel process that directlyconverts one somatic cell into another (a process known astransdifferentiation) (150,151). Functional reprogramming ofa somatic fibroblast cell using a nuclear and cytoplasmic extractderived from another somatic cell type (T-cells) was demon-strated by nuclear uptake and assembly of transcription fac-tors, induction of activity of a chromatin remodeling complex,changes in chromatin composition, and activation of lymphoidcell-specific genes. The reprogrammed cells expressed surfacemolecules specific to T-cells and exhibited apparently normalregulatory functions. In vitro cell reprogramming may alsoallow an examination of the mechanisms of nuclear repro-gramming. Its applicability remains limited by the largeamount of cell extract needed to reprogram a single cell; it isestimated that the extract from 300 cells is required to initiatetransdifferentiation in a single cell. This approach may be use-ful in the identification and characterization of new moleculesinvolved in the remodeling/reprogramming process.

ConclusionsAlthough therapeutic cloning for the purpose of creating

patient-specific stem cells has considerable promise, its real-ization depends on the development of robust SCNT tech-nologies utilizing human oocytes. Since the birth of the firstmammalian clone almost 10 yr ago, the technique has beensuccessfully accomplished in a wide range of mammalianspecies. Although the rapid rate of progress in this area is cer-tainly impressive, both cloned embryos and offspring exhibit

variations in regulation of gene expression as a consequenceof the epigenetic interplay between the original somatic celland its new embryonic environment. As a result, only a smallpercent of cloned embryos result in viable offspring; however,surviving clones appear to have equivalent physiological andreproductive capacities when compared with their nonclonedcounterparts. As a result of these studies, a considerable bodyof work has been accumulated on the technical, biological, andmolecular aspect of nuclear transfer in mammals. Key factorsin the success of SCNT appear consistent across mammals andinclude cell type, degree of cell-cycle synchrony, effectivenessof parthenogenetic activation, and in vitro culture conditions.

Recent developments have focused on events surroundingreprogramming and the initiation of embryonic developmentin an effort to more closely replicate the well-orchestratedevents observed in more developmentally competent IVF andin vivo embryos. It appears that when fully optimized, in vitrodevelopment rates of cloned and parthenogenetic embryosmay approach those of in vitro fertilized embryos, suggestingan important gauge of SCNT proficiency in any given species.Of increasing relevance to human therapeutic cloning, is thatthe efficiency of deriving ESC lines from cloned embryos issubstantially more efficient than the production of viable off-spring. Moreover, ES lines derived from cloned blastocystsappear to share identical characteristics to those derived fromIVF embryos.

However, it is possible that a greater proportion of ESC linesmight have a high rate of epigenetic errors because statisti-cally many of the original embryos would not have resultedin viable animals. Nonetheless, recent reports (152) showingtranscriptional profiles consistent with normal developmentof ES cell lines derived from cloned and fertilized mouse blas-tocysts, were functionally indistinguishable and had identicaltherapeutic potential. The derivation of ESC from clonedembryos appears to rigorously select for those immortal cellsthat have erased the “epigenetic memory” of the donor nucleus,in contrast to aberrant cloning phenotypes observed in theembryonic and fetal development of cloned animals (152).

Progress in SCNT in humans has been disappointing andis likely to be constrained by the ethical considerationsinvolved in the procurement of high-quality oocytes. However,the enormous potential of patient-specific and disease-specificESC lines has reinvigorated the research effort in human ther-apeutic cloning. With research burgeoning in this area, it seemsit is only a matter of time before it will be successfully accom-plished. The full realization of the therapeutic utility of NTSCis likely to be dependent on the ability to establish human ESClines in defined conditions that allow the rapid multiplicationof a purified population of an undifferentiated homogenouscell type, whereas avoiding contamination by animal prod-ucts and pathogens.

Advances in human SCNT for the purpose of ESC linederivation are likely to result from a careful examination ofongoing research undertaken for mammalian reproductivecloning and other synergistic technologies associated withreprogramming. Hopefully, the use of this information willallow a higher level of efficiency for human therapeuticcloning and assist in developing guiding principles that willfacilitate the accomplishment of autologous ESC lines forcell-based therapies (153–156). In the meantime, it is apparent

274 _________________________________________________________________________________________________ French et al.


that parthenogenetically activated oocytes are capable ofproducing nonembryonic pluripotential stem cells and thissource of stem cells for therapeutic application should beexplored in detail.

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