PERSPECTIVE - ALGORISM

1 N engl j med 351;27 30, 2004 2787 PERSPECTIVE Human Cloning The Science and ethics of Nuclear Transplantation Rudolf Jaenisch, In addition to the moral argument against the useof somatic-cell nuclear transfer for the creation ofa child ( reproductive cloning ), there are over-whelming scientific reasons to oppose this contrast, many believe that the practice of somat-ic-cell nuclear transfer with the goal of generatingan embryonic stem-cell line (sometimes referred toas therapeutic cloning ) is justified, because itholds the promise of yielding new ways of studyingand treating a number of diseases. Once isolatedfrom a patient, an embryonic stem cell thus derivedwould be customized to the needs of the patientwho had served as the nuclear donor and thus wouldobviate the need for immunosuppressive treatmentas part of a therapeutic application. In addition, be-cause embryonic stem cells can generate most, ifnot all, types of cells in vitro, a stem cell isolatedfrom a patient with a complex genetic disease couldbe used to study the pathogenesis of the disease inculture.

2 In Figure 1, the generation of a mouse bysexual reproduction is juxtaposed with its genera-tion by nuclear cloning and the derivation of an em-bryonic stem-cell line by means of nuclear transferfrom a patient s contrast to an embryo derived by in vitro fer-tilization, a cloned embryo has little, if any, potentialto develop into a normal human being. By circum-venting the normal processes of gametogenesis andfertilization, nuclear cloning prevents the proper re-programming of the clone s genome (described be-low), which is a prerequisite for the development ofan embryo into a normal organism. It is unlikelythat these biologic barriers to normal developmentcan be overcome in the foreseeable future. However,embryonic stem cells derived from a cloned embryoare functionally indistinguishable from those thathave been generated from embryos derived throughin vitro fertilization. Both have an identical potentialto serve as a source for cells that may prove usefulfor research or therapy.

3 1 Most cloned mammals derived by nuclear trans-fer die during gestation, and those that survive tobirth frequently have the large offspring syndrome,a neonatal phenotype characterized by respiratoryand metabolic abnormalities and an enlarged, dys-functional placenta. In order for a donor nucleus tosupport development into a clone, it must be re-programmed to a state compatible with embryon-ic development. Inadequate reprogramming of thedonor nucleus is most likely the principal reason forthe developmental failure of clones. The transferrednucleus must properly activate genes that are impor-tant for early embryonic development and must alsosuppress genes associated with differentiation thathave been transcribed in the original donor , gene-expression analyses indicate that4 to 5 percent of the overall genome and 30 to 50percent of imprinted genes (described below) arenot correctly expressed in tissues of newborn clonedmice. 2 These data represent strong molecular evi-dence that cloned animals, even if they survive tobirth, have serious gene-expression , as cloned mice age, severe pathologicalalterations in multiple organs and major metabolicdisturbances that were not apparent at younger agesbecome manifest.

4 A case in point is Dolly the sheep,the first mammal cloned from a somatic cell, whichappeared healthy at a young age but died premature-ly with numerous pathological abnormalities. Thesefindings suggest that clones that survive to birthmerely represent the least abnormal animals: sub-tle abnormalities that originate in faulty reprogram-ming may simply not be severe enough to interferewith their survival. Indeed, given the available evi-dence, it may be exceedingly difficult, if not impossi-ble, to generate healthy cloned animals or is often argued that the technical problems as-sociated with producing normal cloned mammalswill be solved by scientific progress in the foresee-able future. But some of these problems may wellprove to be insurmountable. Dr. Jaenisch is a member of the Whitehead Insti-tute for Biomedical Research and a professor of bi-ology at the Massachusetts Institute of Technology, 2004 Massachusetts Medical Society. All rights reserved. n engl j med 351;27 30, 2004 2788 PERSPECTIVE A principal biologic barrier to the creation of anormal animal through cloning is the epigeneticdifference between the chromosomes inheritedfrom the mother (the maternal genome) and thoseinherited from the father (the paternal genome).

5 Forexample, parent-specific methylation marks are re-sponsible for the expression of so-called imprintedgenes and cause only one copy of such a gene, de-rived from either the sperm or the oocyte, to be ac-tive, whereas the other allele is inactive. The mono-allelic expression of imprinted genes is crucial fornormal fetal development. When the genomes ofthe sperm and the oocyte are combined at fertiliza-tion, the parent-specific marks established duringoogenesis and spermatogenesis persist in the ge-nome of the zygote (see Figure 2). Within hours Figure 1. Comparison of Normal Development with Reproductive Cloning and the Derivation of Embryonic Stem Cells through Nuclear Transfer ( Therapeutic Cloning ). During normal development (left), a haploid (1n) sperm cell fertilizes a haploid oocyte to form a diploid (2n) zygote that undergoes cleavage to become a blastocyst embryo. The blastocyst is implanted in the uterus and ultimately develops into a newborn animal.

6 During reproduc-tive cloning (center), the diploid nucleus of an adult donor cell is introduced into an enucleated oocyte recipient, which, after artificial acti-vation, divides into a cloned (nuclear-transfer) blastocyst. On transfer into surrogate mothers, a few of these blastocysts will develop into newborn clones, and most will be abnormal. In contrast, the derivation of embryonic stem cells through nuclear transfer (right) requires the explantation of cloned blastocysts in culture in order to derive an embryonic stem-cell line that can be differentiated in vitro, potentially into any type of cell that occurs in the body, to be used in research or for therapeutic vitrofertilizationReproductive cloningDerivation of embryonic stem cellsNormal Development or in Vitro FertilizationNuclear TransferSperm (1n)Oocyte (1n)Zygote (2n)BlastocystImplantationin uterusTransfer into uterusof surrogate motherAdult mouseCloned mouseNeuronsAdult cell (2n)Patient's cell (2n)EnucleatedoocyteEnucleatedoocyteNucl eartransferNucleartransferNuclear-transf erembryo (2n)Nuclear-transferembryo (2n)Nuclear-transferblastocystNuclear-tr ansferblastocystEmbryonicstem cells(derived fromblastocyst innercell mass)

7 In vitrodifferentiationBlood cellsMuscle cells Human Cloning The Science and ethics of Nuclear TransplantationCopyright 2004 Massachusetts Medical Society. All rights reserved. n engl j med 351;27 30, 2004 2789 PERSPECTIVE after fertilization, most of the global methylationmarks, except those of imprinted genes, are strippedfrom the sperm genome; the oocyte genome, how-ever, is in a chromatin state (inaccessible to the re-programming factors) that renders it resistant tothis process of active contrast, after nuclear transfer, the epigeneticdifferences established during gametogenesis aresubject to erasure, because both parental genomesof the somatic donor cell not just the sperm ge-nome, as in fertilization are introduced into theoocyte from the outside and are thus equally ex-posed to the reprogramming activity of the oocytecytoplasm. Therefore, in cloned animals, imprintedgenes should be particularly vulnerable to inappro-priate methylation, which causes abnormal expres-sion a prediction that has, as noted above, beenverified experimentally.

8 For cloning to be made safe,the two parental genomes of a somatic donor cellwould need to be physically separated and individu-ally treated in oocyte-appropriate and sperm-appropriate ways. At present, it seems that this isthe only rational approach to guaranteeing the cre-ation of the epigenetic differences that are nor-mally established during gametogenesis. Such anapproach is beyond our present abilities, implyingthat serious biologic barriers (rather than mere tech-nical problems) hinder faithful reprogramming af-ter nuclear transfer and thus preclude the use of nu-clear cloning as a safe reproductive generation of embryonic stem cells by nu-clear transplantation for use in therapeutic applica-tions is another story. Embryonic stem cells are de-rived from the portion of the blastocyst termed theinner cell mass. The cells of the inner cell mass ex-press key embryonic genes such as the transcrip-tion factor Oct-4. After explantation in culture, thesecells extinguish Oct-4 expression and cease to pro-liferate.

9 Only one or a few of the cells derived fromthe inner cell mass will eventually express Oct-4 Figure 2. Parental Epigenetic Differences in Normal and Cloned Animals. The genomes of oocyte and sperm are differentially methylated (epigenetically marked ) during gametogenesis and are different in the zygote when they are combined at fertilization. Immediately after fertilization, the paternal genome (derived from the sperm) is actively demethylated, whereas the maternal genome is only partially demethylated during the next few days of cleavage; this is because the oocyte genome, which has a different chromatin configuration from the sperm genome, is resistant to the active demethylation process imposed on the genome of the sperm by the cytoplasm of the oocyte. Thus, the methylation marks of the two parental genomes are different at the end of the cleavage process and in the adult. In cloning, a somatic nucleus is transferred into the enucleated oocyte, and both parental genomes are exposed to the active demethylating activity of the cytoplasm of the oocyte.

10 Therefore, the parent-specific epigenetic dif-ferences between the two genomes tend to become erased, causing widespread dysregulation of imprinted genes. Methylated sequences are depicted as solid circles, and unmethylated sequences as open DevelopmentNuclear-Transfer CloningGametogenesisFertilizationCleavag eDifferent marks onparental genomesParental marksequalizedEggEnucleated eggCloned embryoCloned adultAdultEnucleationHoursDaysYearsNucle artransferDemethylation of paternal genome Human Cloning The Science and ethics of Nuclear TransplantationCopyright 2004 Massachusetts Medical Society. All rights reserved. n engl j med 351;27 30, 2004 2790 PERSPECTIVE again, and these few cells will resume rapid prolifer-ation, yielding the cell populations that we call em-bryonic stem cells. 1 Thus, the propagation of blas-tocyst cells in vitro results in a rare population ofsurviving cells in which, as a consequence of the se-lection for proliferation in culture, the epigeneticmemory of the donor nucleus has been erased (seeFigure 3).