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



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作者：Vanessa J. Halla, Petra Stojkovicb, Miodrag Stojkovicb作者单位：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 # V3 c* Q$ _' X' W1 X' P0 c: Q
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      【摘要】
   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.
      【关键词】 Clinical stem cell transplantation Reprogramming Human embryonic stem cells Cloning) F8 [* j+ u' C$ t: c& p4 J8 V4 T6 Q
   INTRODUCTION8 B- r; P3 D3 S7 t. N2 [
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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 . 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. 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.; Q) P6 V- u/ A; K9 K- K4 g
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Studying NT-ESCs in Animal Models  h6 D' l, a; O) D' e
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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 .% N: M/ i, Q) w% F2 q
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Potential of Differentiated NT-ESCs to Treat Disease* E% C/ ]6 z- l6 Y4 H; F
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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  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.) C7 l, x% d5 B3 C# V# I/ \

Progress in Production of Human and Primate NT-ESC Lines
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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 . 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.

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 . 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( ?  i( T# K' V2 L3 ]
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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 ; 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.4 _' G$ w+ Z5 @; C% G
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Ethical and Societal Concerns) L# d( V/ |" o9 I# Q! c

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¨C$2,000 from consenting women or from volunteer programs where the woman is reimbursed for the clinical procedures in infertility treatments . 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.6 G3 d2 H% _8 `# G+ ]

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 .  z. f: |% N) h* `- B! Z
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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 , 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.
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Perspectives on Current Legislation2 K! `2 B$ q# l7 ?: ^

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 , 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.: S( D" U$ p: G8 F

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)6 a( {- h8 R+ z% f& t* c
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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 . Currently, legislation pertaining to the use of ESCs and hNT is determined by individual states.4 I' u/ l: ]8 S6 Y

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.  ?3 K( s3 b( g1 T( w6 B( l) Y

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 .4 [7 ^" e) f  V5 r, S

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 .  q9 k" {$ v( h9 _/ a

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.% u, x5 a7 Z; L  k, M7 s

Mitochondrial Heteroplasmy and Possible Role in Immunorejection, L- ^5 M* ~) N  K5 h
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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 .
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Figure 3. Possible mechanism of acute graft rejection due to mitochondrial heteroplasmy following cell transplantation. Loss of 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.- s0 ?- i! i  _- A, B! K4 @
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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 . 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& ~1 g3 \+ n( z* b

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 . 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.' e( G0 |2 k4 D; e- H3 C2 J5 O

Epigenetic defects may also result in altered telomere lengths in reproductive NT offspring , 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
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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 .
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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 ; however, developing stem cell banks comprised of histocompatible NT-ESCs may be even more advantageous.# h8 X- P* v5 o, }) X. [. u

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 . 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 .

CONCLUSION9 ?6 ~0 ?1 t0 |3 |5 X: ?7 R

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.& a8 w1 L8 I! \* E1 x% C3 ?& S

DISCLOSURES
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The authors indicate no potential conflicts of interest.

ACKNOWLEDGMENTS4 I3 t) J$ k! C& u  j; H

We thank Simon Foster for the compilation of the illustrations in this manuscript., U9 k2 L9 }6 I: u, [
      【参考文献】

Kind A, Colman A. Therapeutic cloning: Needs and prospects. Semin Cell Dev Biol 1999;10:279¨C286.
, O/ D: [, P9 L" u
Lanza RP, Cibelli JB, West MD. Human therapeutic cloning. Nat Med 1999;5:975¨C977.8 v4 y' ^- q5 a2 W, V

Trounson A. Nuclear transfer in human medicine and animal breeding. Reprod Fertil Dev 2001;13:31¨C39.

Doss MX, Koehler CI, Gissel C, Hescheler J, Sachinidis A. Embryonic stem cells: A promising tool for cell replacement therapy. J Cell Mol Med 2004;8:465¨C473., \  W: |' _. R% H, B# L

Hoffman LM, Carpenter MK. Characterization and culture of human embryonic stem cells. Nat Biotechnol 2005;23:699¨C708.$ a1 f0 l7 K, Q/ A$ K  h# m0 y' w

Vats A, Tolley NS, Bishop AE et al. Embryonic stem cells and tissue engineering: Delivering stem cells to the clinic. J R Soc Med 2005;98:346¨C350.
0 z0 P: I" o% @2 P0 R' g8 L
Munsie MJ, Michalska AE, O¡¯Brien CM et al. Isolation of pluripotent embryonic stem cells from reprogrammed adult mouse somatic cell nuclei. Curr Biol 2000;10:989¨C992.4 Z5 Y8 C; p6 ~6 H; m
) P% F4 h" ]2 e& k' o& s
Kawase E, Yamazaki Y, Yagi T et al. Mouse embryonic stem (ES) cell lines established from neuronal cell-derived cloned blastocysts. Genesis 2000;28:156¨C163.: A+ S2 t: z% \0 N2 G* y

Wakayama T, Tabar V, Rodriguez I et al. Differentiation of embryonic stem cell lines generated from adult somatic cells by nuclear transfer. Science 2001;292:740¨C743.; M# G( M: B( [5 C/ N5 D

Rideout WM 3rd, Hochedlinger K, Kyba M et al. Correction of a genetic defect by nuclear transplantation and combined cell and gene therapy. Cell 2002;109:17¨C27.7 @5 h- k% ]3 L. {3 J% H) @
  Z8 N# x9 q7 ]! q; J
Barberi T, Klivenyi P, Calingasan NY et al. Neural subtype specification of fertilization and nuclear transfer embryonic stem cells and application in parkinsonian mice. Nat Biotechnol 2003;21:1200¨C1207.0 e8 G* n$ N8 Q
/ ~! H$ l% m% k; i
Down JD, White-Scharf ME. Reprogramming immune responses: Enabling cellular therapies and regenerative medicine. STEM CELLS 2003;21:21¨C32.

Strom TB, Field LJ, Ruediger M. Allogeneic stem cells, clinical transplantation and the origins of regenerative medicine. Curr Opin Immunol 2002;14:601¨C605.

Takagi Y, Takahashi J, Saiki H et al. Dopaminergic neurons generated from monkey embryonic stem cells function in a Parkinson primate model. J Clin Invest 2005;115:102¨C109.

Faulkner J, Keirstead HS. Human embryonic stem cell-derived oligodendrocyte progenitors for the treatment of spinal cord injury. Transpl Immunol 2005;15:131¨C142.
4 Q/ U' f# J* P4 {
Keirstead HS, Nistor G, Bernal G et al. Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury. J Neurosci 2005;25:4694¨C4705., r3 o" r1 h9 P0 ]" ]" e; b
. f9 N0 T/ [, B2 z. y9 h
Kimura H, Yoshikawa M, Matsuda R et al. Transplantation of embryonic stem cell-derived neural stem cells for spinal cord injury in adult mice. Neurol Res 2005;27:812¨C819.$ I  Q" a) R2 f: b5 f

Menard C, Hagege AA, Agbulut O et al. Transplantation of cardiac-committed mouse embryonic stem cells to infarcted sheep myocardium: A preclinical study. Lancet 2005;366:1005¨C1012.
4 r# M/ a) Z  A7 }: `8 ~8 X  \
Stojkovic M, Stojkovic P, Leary C et al. Derivation of a human blastocyst after heterologous nuclear transfer to donated oocytes. Reprod Biomed Online 2005;11:226¨C231.

Lavoir M-C, Weier J, Conaghan J et al. Poor development of human nuclear transfer embryos using failed fertilized oocytes. Reprod Biomed Online 2005;11:740¨C744.( l, s9 p2 d! ]! t( D# `2 c
% r' Z: f2 i! Q! P, z+ F
Simerly C, Navara C, Hyun SH et al. Embryogenesis and blastocyst development after somatic cell nuclear transfer in nonhuman primates: Overcoming defects caused by meiotic spindle extraction. Dev Biol 2004;276:237¨C252.
5 p% X! B! @  K' g5 U: x0 S
Simerly C, Dominko T, Navara C et al. Molecular correlates of primate nuclear transfer failures. Science 2003;300:297.3 k* e: ^& A3 j4 R# I# g

Meng L, Ely JJ, Stouffer RL et al. Rhesus monkeys produced by nuclear transfer. Biol Reprod 1997;57:454¨C459.
3 g. w1 G3 b8 t3 Z
Ludwig TE, Levenstein ME, Jones JM et al. Derivation of human embryonic stem cells in defined conditions. Nat Biotechnol 2006;24:185¨C187.% {1 u$ ?- b5 H' B4 b6 V: Q
; y: [! A$ Q6 N. A. o" ?3 y  X
Takagi Y, Nishimura M, Morizane A et al. Survival and differentiation of neural progenitor cells derived from embryonic stem cells and transplanted into ischemic brain. J Neurosurg 2005;103:304¨C310.( q% ~5 H3 c7 Z
2 w7 e& T: g, H, Y2 Z; Q
Ruschenschmidt C, Koch PG, Brustle O et al. Functional properties of ES cell-derived neurons engrafted into the hippocampus of adult normal and chronically epileptic rats. Epilepsia 2005;46 (suppl 5):174¨C183.4 m' o5 H- I% \( o! H, B
: Q" \. H! H1 f- m* ^
Fujikawa T, Oh SH, Pi L et al. Teratoma formation leads to failure of treatment for type I diabetes using embryonic stem cell-derived insulin-producing cells. Am J Pathol 2005;166:1781¨C1791.

Mombaerts P. Therapeutic cloning in the mouse. Proc Natl Acad Sci U S A 2003;100 (suppl 1):11924¨C11925.

Blyth E. Subsidized IVF. The development of ¡®egg sharing¡¯ in the United Kingdom. Hum Reprod 2002;17:3254¨C3259.

Heng BC. Egg-sharing in return for subsidized fertility treatment¨Can ethically justifiable and practical solution to overcome the shortage of donor oocytes for therapeutic cloning. Med Hypotheses 2005;65:999¨C1000.9 M1 c5 X/ H: X) C. j5 x: d
7 a- m- f8 ?* \2 J8 L5 x. v* a, ^7 E
Couzin J. Stem cells. Another route to oocytes? Science 2005;309:1983.4 L3 Z+ h' n/ R' h$ f
6 k4 z4 B' ^0 y8 v2 i
Hubner K, Fuhrmann G, Christenson LK et al. Derivation of oocytes from mouse embryonic stem cells. Science 2003;300:1251¨C1256.
6 n3 e$ F4 Y/ @8 j; P9 u
Kehler J, Hubner K, Garrett S et al. Generating oocytes and sperm from embryonic stem cells. Semin Reprod Med 2005;23:222¨C233.
& R1 b  q7 O2 O4 q" s
Daar AS, Bhatt A, Court E et al. Stem cell research and transplantation: Science leading ethics. Transplant Proc 2004;36:2504¨C2506.
+ E; m" K% J# S$ b; T, u0 |
Schenker JG. Assisted reproductive practice: Religious perspectives. Reprod Biomed Online 2005;10:310¨C319.
% O  o' Q/ I! v2 N+ S6 k
Brun RB. Cloning humans? Current science, current views, and a perspective from Christianity. Differentiation 2002;69:184¨C187.0 J0 g) X' E% ]" J( A) H
' n6 w2 M/ u: S* k
Charter of Fundamental Rights of the European Union. Available at http://www.europarl.europa.eu/comparl/libe/elsg/charter/default_en.htm Accessed April 2, 2006.

Wertz DC. Embryo and stem cell research in the United States: History and politics. Gene Ther 2002;9:674¨C678.( R* J) X( {) x! n

Childress JF. Human stem cell research: Some controversies in bioethics and public policy. Blood Cells Mol Dis 2004;32:100¨C105.

Check E. Stem-cell research: The rocky road to success. Nature 2005;437:185¨C186.: ^4 c$ _! m% T5 w0 l

Meissner A, Jaenisch R. Generation of nuclear transfer-derived pluripotent ES cells from cloned Cdx2-deficient blastocysts. Nature 2006;439:212¨C215.

St John JC, Schatten G. Paternal mitochondrial DNA transmission during nonhuman primate nuclear transfer. Genetics 2004;167:897¨C905.& O3 g4 Z  {( V- Q. ^# L
0 L5 m& m5 r& v8 u8 ^7 H
Steinborn R, Schinogl P, Zakhartchenko V et al. Mitochondrial DNA heteroplasmy in cloned cattle produced by fetal and adult cell cloning. Nat Genet 2000;25:255¨C257.

Hiendleder S, Zakhartchenko V, Wenigerkind H et al. Heteroplasmy in bovine fetuses produced by intra- and inter-subspecific somatic cell nuclear transfer: Neutral segregation of nuclear donor mitochondrial DNA in various tissues and evidence for recipient cow mitochondria in fetal blood. Biol Reprod 2003;68:159¨C166.' p" F2 N+ N* V1 S% k1 n

Takeda K, Takahashi S, Onishi A et al. Dominant distribution of mitochondrial DNA from recipient oocytes in bovine embryos and offspring after nuclear transfer. J Reprod Fertil 1999;116:253¨C259.% I, v) o  f" O: a$ [3 m
7 q; B& [/ C% Z; x% w3 C8 k4 s" I
Steinborn R, Zakhartchenko V, Wolf E et al. Non-balanced mix of mitochondrial DNA in cloned cattle produced by cytoplast-blastomere fusion. FEBS Lett 1998;426:357¨C361.4 r4 m/ s& C+ F, E
# U7 m8 D+ N, }; [# p/ l7 Q
Hiendleder S, Schmutz SM, Erhardt G et al. Transmitochondrial differences and varying levels of heteroplasmy in nuclear transfer cloned cattle. Mol Reprod Dev 1999;54:24¨C31.+ c5 K) p$ M9 K+ r, z+ E

Evans MJ, Gurer C, Loike JD et al. Mitochondrial DNA genotypes in nuclear transfer-derived cloned sheep. Nat Genet 1999;23:90¨C93.5 k8 @* z5 g# E- n2 }' b  H
1 s; s: l# |/ g- G; X  ?& m
Brenner CA, Kubisch HM, Pierce KE. Role of the mitochondrial genome in assisted reproductive technologies and embryonic stem cell-based therapeutic cloning. Reprod Fertil Dev 2004;16:743¨C751.

Lanza RP, Chung HY, Yoo JJ et al. Generation of histocompatible tissues using nuclear transplantation. Nat Biotechnol 2002;20:689¨C696.
& o6 z' i: B9 [; |0 n; C
Raisky O, Gomez L, Chalabreysse L et al. Mitochondrial permeability transition in cardiomyocyte apoptosis during acute graft rejection. Am J Transplant 2004;4:1071¨C1078.& y5 F2 p' U" T; y/ K- j0 R
! p/ {" u4 ^7 j' y5 d4 F
Kristal BS, Stavrovskaya IG, Narayanan MV et al. The mitochondrial permeability transition as a target for neuroprotection. J Bioenerg Biomembr 2004;36:309¨C312.

Vats A, Bielby RC, Tolley NS et al. Stem cells. Lancet 2005;366:592¨C602.4 v3 p$ \/ B2 W$ t% |; v
: D7 ]1 {# {1 W/ k6 t# I8 k- D
Dabhi VM, Lindahl KF. MtDNA-encoded histocompatibility antigens. Methods Enzymol 1995;260:466¨C485.9 W8 t9 S4 A  F9 x# U2 k

Fischer Lindahl K, Hermel E, Loveland BE et al. Maternally transmitted antigen of mice: A model transplantation antigen. Annu Rev Immunol 1991;9:351¨C372.

Zavazava N, Kabelitz D. Alloreactivity and apoptosis in graft rejection and transplantation tolerance. J Leukoc Biol 2000;68:167¨C174.

Zoratti M, Szabo I. The mitochondrial permeability transition. Biochim Biophys Acta 1995;1241:139¨C176.5 X) d) i& F, s4 b9 K+ @, B
- V5 d% b0 j4 q$ g/ K7 O( e# @
Kahan BD. Cyclosporine: A revolution in transplantation. Transplant Proc 1999;31(1¨C2A):14S¨C15S.+ H& y2 k, d  a; w( J& k

Streilein JW. Peripheral tolerance induction: Lessons from immune privileged sites and tissues. Transplant Proc 1996;28:2066¨C2070.

Taylor RW, Turnbull DM. Mitochondrial DNA mutations in human disease. Nat Rev Genet 2005;6:389¨C402.2 ]1 G2 H, x- [2 t/ ]9 v! ?# t
8 f/ i4 x' @7 B; [( @% i: o: Y1 u
Dimauro S, Davidzon G. Mitochondrial DNA and disease. Ann Med 2005;37:222¨C232.

Chinnery PF, Thorburn DR, Samuels DC et al. The inheritance of mitochondrial DNA heteroplasmy: Random drift, selection or both? Trends Genet 2000;16:500¨C505.

Morgan HD, Santos F, Green K et al. Epigenetic reprogramming in mammals. Hum Mol Genet 2005;14(spec 1):R47¨CR58.: D3 l) `; Q! ?- m6 E. L  R( @

Humpherys D, Eggan K, Akutsu H et al. Abnormal gene expression in cloned mice derived from embryonic stem cell and cumulus cell nuclei. Proc Natl Acad Sci U S A 2002;99:12889¨C12894.

Jouneau A, Renard JP. Reprogramming in nuclear transfer. Curr Opin Genet Dev 2003;13:486¨C491.

Niemann H, Wrenzycki C, Lucas-Hahn A et al. Gene expression patterns in bovine in vitro-produced and nuclear transfer-derived embryos and their implications for early development. Cloning Stem Cells 2002;4:29¨C38.

Wrenzycki C, Herrmann D, Lucas-Hahn A et al. Gene expression patterns in in vitro-produced and somatic nuclear transfer-derived preimplantation bovine embryos: Relationship to the large offspring syndrome? Anim Reprod Sci 2004;82¨C83:593¨C603.' O8 U; ^2 P. x) s
( h6 E" N5 o' h
Hall VJ, Ruddock NT, French AJ. Expression profiling of genes crucial for placental and preimplantation development in bovine in vivo, in vitro, and nuclear transfer blastocysts. Mol Reprod Dev 2005;72:16¨C24.

Dean W, Santos F, Stojkovic M et al. Conservation of methylation reprogramming in mammalian development: Aberrant reprogramming in cloned embryos. Proc Natl Acad Sci U S A 2001;98:13734¨C13738.

Humpherys D, Eggan K, Akutsu H et al. Epigenetic instability in ES cells and cloned mice. Science 2001;293:95¨C97.( ?  z6 W, [. b& Q  G1 Q0 y$ w% J
: ]0 z9 j3 ^6 S( }- q0 w5 U5 Y
Santos F, Zakhartchenko V, Stojkovic M et al. Epigenetic marking correlates with developmental potential in cloned bovine preimplantation embryos. Curr Biol 2003;13:1116¨C1121.

Xue F, Tian XC, Du F et al. Aberrant patterns of X chromosome inactivation in bovine clones. Nat Genet 2002;31:216¨C220.
# h" k% u# F3 k7 f9 y4 u
Eggan K, Akutsu H, Hochedlinger K, Rideout W 3rd et al. X-chromosome inactivation in cloned mouse embryos. Science 2000;290:1578¨C1581.% [3 f  V- \6 t

Mann MR, Chung YG, Nolen LD et al. Disruption of imprinted gene methylation and expression in cloned preimplantation stage mouse embryos. Biol Reprod 2003;69:902¨C914.

Skottman H, Mikkola M, Lundin K et al. Gene expression signatures of seven individual human embryonic stem cell lines. STEM CELLS 2005;23:1343¨C1356.! q  o8 ~3 L* X. I2 |7 E$ p4 R+ l
: x& W3 m# ?1 Z3 A0 O  Q
Yang X, Tian XC. Cloning adult animals¨Cwhat is the genetic age of the clones? Cloning 2000;2:123¨C128.

Shiels PG, Kind AJ, Campbell KH et al. Analysis of telomere length in Dolly, a sheep derived by nuclear transfer. Cloning 1999;1:119¨C125.
- w: T6 D& p* c0 h& n! l
Jiang L, Carter DB, Xu J et al. Telomere lengths in cloned transgenic pigs. Biol Reprod 2004;70:1589¨C1593.

Jeon HY, Hyun SH, Lee GS et al. The analysis of telomere length and telomerase activity in cloned pigs and cows. Mol Reprod Dev 2005;71:315¨C320.
& C9 I8 {- K7 _+ c$ h, w2 X7 k
Miura T, Mattson MP, Rao MS. Cellular lifespan and senescence signaling in embryonic stem cells. Aging Cell 2004;3:333¨C343.
/ l" Y. o) b% T% R: E4 r  A8 Z
Young HE, Duplaa C, Katz R et al. Adult-derived stem cells and their potential for use in tissue repair and molecular medicine. J Cell Mol Med 2005;9:753¨C769.  [3 `( Z7 f1 v  m. [

Rice CM, Scolding NJ. Adult stem cells¨Creprogramming neurological repair? Lancet 2004;364:193¨C199.

Rubio D, Garcia-Castro J, Martin MC et al. Spontaneous human adult stem cell transformation. Cancer Res 2005;65:3035¨C3039.
  F& B& R8 N: Q9 ^; f6 y) y
Baksh D, Song L, Tuan RS. Adult mesenchymal stem cells: Characterization, differentiation, and application in cell and gene therapy. J Cell Mol Med 2004;8:301¨C316.  s! Z5 E& f1 y0 I- ^( k
" B. ^) M+ _% G, b2 l
Hassan HT, El-Sheemy M. Adult bone-marrow stem cells and their potential in medicine. J R Soc Med 2004;97:465¨C471.
  Z8 I) c* c! j7 k$ H
Drukker M, Katz G, Urbach A et al. Characterization of the expression of MHC proteins in human embryonic stem cells. Proc Natl Acad Sci U S A 2002;99:9864¨C9869.

Westphal CH, Leder P. Transposon-generated ¡®knock-out¡¯ and ¡®knock-in¡¯ gene-targeting constructs for use in mice. Curr Biol 1997;7:530¨C533.; I7 n$ R4 [- _9 ^! y

Martinez OM, Krams SM. Involvement of Fas-Fas ligand interactions in graft rejection. Int Rev Immunol 1999;18:527¨C546.8 \2 r) h: b2 L' ^+ {$ _+ e( t

Guillonneau C, Aubry V, Renaudin K et al. Inhibition of chronic rejection and development of tolerogenic T cells after ICOS-ICOSL and CD40-CD40L co-stimulation blockade. Transplantation 2005;80:255¨C263.

Taylor CJ, Bolton EM, Pocock S et al. Banking on human embryonic stem cells: Estimating the number of donor cell lines needed for HLA matching. Lancet 2005;366:2019¨C2025.
4 g0 {/ P7 |# h# K+ a
Cowan CA, Atienza J, Melton DA et al. Nuclear reprogramming of somatic cells after fusion with human embryonic stem cells. Science 2005;309:1369¨C1373.3 \/ f1 }4 r" m- \+ t

Ambrosi DJ, Rasmussen TP. Reprogramming mediated by stem cell fusion. J Cell Mol Med 2005;9:320¨C330.
8 D  k) y. n( `2 L% h* @- h
Ringertz N, Savage RE. Cell Hybrids. New York: Academic Press, 1976;.  r: `+ X1 V; E9 c) t
/ e0 d+ S2 ~3 P
Weimann JM, Johansson CB, Trejo A et al. Stable reprogrammed heterokaryons form spontaneously in Purkinje neurons after bone marrow transplant. Nat Cell Biol 2003;5:959¨C966.: d/ w( ]2 @% c7 `$ Y

Wang X, Willenbring H, Akkari Y et al. Cell fusion is the principal source of bone-marrow-derived hepatocytes. Nature 2003;422:897¨C901.
8 A1 y, t4 q7 Z2 O1 r2 P
Vassilopoulos G, Wang PR, Russell DW. Transplanted bone marrow regenerates liver by cell fusion. Nature 2003;422:901¨C904.

Kiessling AA. Eggs alone. Nature 2005;434:145.0 ?/ A5 A% B5 e8 Y1 s3 D
4 w+ H- n' a! d. s3 B7 M: q, Y# E
Vrana KE, Hipp JD, Goss AM et al. Nonhuman primate parthenogenetic stem cells. Proc Natl Acad Sci U S A 2003;100 (suppl 1):11911¨C11916.
1 P6 d: v: i4 Q6 C3 r  h# T# v% N2 P
Kotecki M, Reddy PS, Cochran BH. Isolation and characterization of a near-haploid human cell line. Exp Cell Res 1999;252:273¨C280.