Cloning: Definitions And Applications
In this chapter, we address the following questions in our task statement:
What does cloning of animals including humans mean? What are its purposes? How does it differ from stem cell research?
To organize its response to those questions, the panel developed a series of subquestions, which appear as the section headings in the following text.
WHAT IS MEANT BY REPRODUCTIVE CLONING OF ANIMALS INCLUDING HUMANS?
Reproductive cloning is defined as the deliberate production of genetically identical individuals. Each newly produced individual is a clone of the original. Monozygotic (identical) twins are natural clones. Clones contain identical sets of genetic material in the nucleus—the compartment that contains the chromosomes—of every cell in their bodies. Thus, cells from two clones have the same DNA and the same genes in their nuclei.
All cells, including eggs, also contain some DNA in the energy-generating “factories” called mitochondria. These structures are in the cytoplasm, the region of a cell outside the nucleus. Mitochondria contain their own DNA and reproduce independently. True clones have identical DNA in both the nuclei and mitochondria, although the term clones is also used
to refer to individuals that have identical nuclear DNA but different mitochondrial DNA.
HOW IS REPRODUCTIVE CLONING DONE?
Two methods are used to make live-born mammalian clones. Both require implantation of an embryo in a uterus and then a normal period of gestation and birth. However, reproductive human or animal cloning is not defined by the method used to derive the genetically identical embryos suitable for implantation. Techniques not yet developed or described here would nonetheless constitute cloning if they resulted in genetically identical individuals of which at least one were an embryo destined for implantation and birth.
The two methods used for reproductive cloning thus far are as follows:
• Cloning using somatic cell nuclear transfer (SCNT) . This procedure starts with the removal of the chromosomes from an egg to create an enucleated egg. The chromosomes are replaced with a nucleus taken from a somatic (body) cell of the individual or embryo to be cloned. This cell could be obtained directly from the individual, from cells grown in culture, or from frozen tissue. The egg is then stimulated, and in some cases it starts to divide. If that happens, a series of sequential cell divisions leads to the formation of a blastocyst, or preimplantation embryo. The blastocyst is then transferred to the uterus of an animal. The successful implantation of the blastocyst in a uterus can result in its further development, culminating sometimes in the birth of an animal. This animal will be a clone of the individual that was the donor of the nucleus. Its nuclear DNA has been inherited from only one genetic parent.
The number of times that a given individual can be cloned is limited theoretically only by the number of eggs that can be obtained to accept the somatic cell nuclei and the number of females available to receive developing embryos. If the egg used in this procedure is derived from the same individual that donates the transferred somatic nucleus, the result will be an embryo that receives all its genetic material—nuclear and mitochondrial—from a single individual. That will also be true if the egg comes from the nucleus donor’s mother, because mitochondria are inherited maternally. Multiple clones might also be produced by transferring identical nuclei to eggs from a single donor. If the somatic cell nucleus and the egg come from different individuals, they will not be identical to the nuclear donor because the clones will have somewhat different mitochondrial genes [2; 3]
• Cloning by embryo splitting. This procedure begins with in vitro fertilization (IVF): the union outside the woman’s body of a sperm and an
egg to generate a zygote. The zygote (from here onwards also called an embryo) divides into two and then four identical cells. At this stage, the cells can be separated and allowed to develop into separate but identical blastocysts, which can then be implanted in a uterus. The limited developmental potential of the cells means that the procedure cannot be repeated, so embryo splitting can yield only two identical mice and probably no more than four identical humans.
The DNA in embryo splitting is contributed by germ cells from two individuals—the mother who contributed the egg and the father who contributed the sperm. Thus, the embryos, like those formed naturally or by standard IVF, have two parents. Their mitochondrial DNA is identical. Because this method of cloning is identical with the natural formation of monozygotic twins and, in rare cases, even quadruplets, it is not discussed in detail in this report.
WILL CLONES LOOK AND BEHAVE EXACTLY THE SAME?
Even if clones are genetically identical with one another, they will not be identical in physical or behavioral characteristics, because DNA is not the only determinant of these characteristics. A pair of clones will experience different environments and nutritional inputs while in the uterus, and they would be expected to be subject to different inputs from their parents, society, and life experience as they grow up. If clones derived from identical nuclear donors and identical mitocondrial donors are born at different times, as is the case when an adult is the donor of the somatic cell nucleus, the environmental and nutritional differences would be expected to be more pronounced than for monozygotic (identical) twins. And even monozygotic twins are not fully identical genetically or epigenetically because mutations, stochastic developmental variations, and varied imprinting effects (parent-specific chemical marks on the DNA) make different contributions to each twin [3; 4].
Additional differences may occur in clones that do not have identical mitochondria. Such clones arise if one individual contributes the nucleus and another the egg—or if nuclei from a single individual are transferred to eggs from multiple donors. The differences might be expected to show up in parts of the body that have high demands for energy—such as muscle, heart, eye, and brain—or in body systems that use mitochondrial control over cell death to determine cell numbers [5; 6].
WHAT ARE THE PURPOSES OF REPRODUCTIVE CLONING?
Cloning of livestock  is a means of replicating an existing favorable combination of traits, such as efficient growth and high milk production,
without the genetic “lottery” and mixing that occur in sexual reproduction. It allows an animal with a particular genetic modification, such as the ability to produce a pharmaceutical in milk, to be replicated more rapidly than does natural mating [7; 8]. Moreover, a genetic modification can be made more easily in cultured cells than in an intact animal, and the modified cell nucleus can be transferred to an enucleated egg to make a clone of the required type. Mammals used in scientific experiments, such as mice, are cloned as part of research aimed at increasing our understanding of fundamental biological mechanisms.
In principle, those people who might wish to produce children through human reproductive cloning  include:
Infertile couples who wish to have a child that is genetically identical with one of them, or with another nucleus donor
Other individuals who wish to have a child that is genetically identical with them, or with another nucleus donor
Parents who have lost a child and wish to have another, genetically identical child
People who need a transplant (for example, of cord blood) to treat their own or their child’s disease and who therefore wish to collect genetically identical tissue from a cloned fetus or newborn.
Possible reasons for undertaking human reproductive cloning have been analyzed according to their degree of justification. For example, in reference 10 it is proposed that human reproductive cloning aimed at establishing a genetic link to a gametically infertile parent would be more justifiable than an attempt by a sexually fertile person aimed at choosing a specific genome.
Transplantable tissue may be available without the need for the birth of a child produced by cloning. For example, embryos produced by in vitro fertilization (IVF) can be typed for transplant suitability, and in the future stem cells produced by nuclear transplantation may allow the production of transplantable tissue.
The alternatives open to infertile individuals are discussed in Chapter 4.
HOW DOES REPRODUCTIVE CLONING DIFFER FROM STEM CELL RESEARCH?
The recent and current work on stem cells that is briefly summarized below and discussed more fully in a recent report from the National Academies entitled Stem Cells and the Future of Regenerative Medicine  is not directly related to human reproductive cloning. However, the use of a
common initial step—called either nuclear transplantation or somatic cell nuclear transfer (SCNT)—has led Congress to consider bills that ban not only human reproductive cloning but also certain areas of stem cell research. Stem cells are cells that have the ability to divide repeatedly and give rise to both specialized cells and more stem cells. Some, such as some blood and brain stem cells, can be derived directly from adults [12-19] and others can be obtained from preimplantation embryos. Stem cells derived from embryos are called embryonic stem cells (ES cells). The above-mentioned report from the National Academies provides a detailed account of the current state of stem cell research .
ES cells are also called pluripotent stem cells because their progeny include all cell types that can be found in a postimplantation embryo, a fetus, and a fully developed organism. They are derived from the inner cell mass of early embryos (blastocysts) [20-23]. The cells in the inner cell mass of a given blastocyst are genetically identical, and each blastocyst yields only a single ES cell line. Stem cells are rarer  and more difficult to find in adults than in preimplantation embryos, and it has proved harder to grow some kinds of adult stem cells into cell lines after isolation [25; 26].
Production of different cells and tissues from ES cells or other stem cells is a subject of current research [11; 27-31]. Production of whole organs other than bone marrow (to be used in bone marrow transplantation) from such cells has not yet been achieved, and its eventual success is uncertain.
Current interest in stem cells arises from their potential for the therapeutic transplantation of particular healthy cells, tissues, and organs into people suffering from a variety of diseases and debilitating disorders. Research with adult stem cells indicates that they may be useful for such purposes, including for tissues other than those from which the cells were derived [12; 14; 17; 18; 25-27; 32-43]. On the basis of current knowledge, it appears unlikely that adults will prove to be a sufficient source of stem cells for all kinds of tissues [11; 44-47]. ES cell lines are of potential interest for transplantation because one cell line can multiply indefinitely and can generate not just one type of specialized cell, but many different types of specialized cells (brain, muscle, and so on) that might be needed for transplants [20; 28; 45; 48; 49]. However, much more research will be needed before the magnitude of the therapeutic potential of either adult stem cells or ES cells will be well understood.
One of the most important questions concerning the therapeutic potential of stem cells is whether the cells, tissues, and perhaps organs derived from them can be transplanted with minimal risk of transplant rejection. Ideally, adult stem cells advantageous for transplantation might be derived from patients themselves. Such cells, or tissues derived from
them, would be genetically identical with the patient’s own and not be rejected by the immune system. However, as previously described, the availability of sufficient adult stem cells and their potential to give rise to a full range of cell and tissue types are uncertain. Moreover, in the case of a disorder that has a genetic origin, a patient’s own adult stem cells would carry the same defect and would have to be grown and genetically modified before they could be used for therapeutic transplantation.
The application of somatic cell nuclear transfer or nuclear transplantation offers an alternative route to obtaining stem cells that could be used for transplantation therapies with a minimal risk of transplant rejection. This procedure—sometimes called therapeutic cloning, research cloning, or nonreproductive cloning, and referred to here as nuclear transplantation to produce stem cells—would be used to generate pluripotent ES cells that are genetically identical with the cells of a transplant recipient . Thus, like adult stem cells, such ES cells should ameliorate the rejection seen with unmatched transplants.
Two types of adult stem cells—stem cells in the blood forming bone marrow and skin stem cells—are the only two stem cell therapies currently in use. But, as noted in the National Academies’ report entitled Stem Cells and the Future of Regenerative Medicine, many questions remain before the potential of other adult stem cells can be accurately assessed . Few studies on adult stem cells have sufficiently defined the stem cell’s potential by starting from a single, isolated cell, or defined the necessary cellular environment for correct differentiation or the factors controlling the efficiency with which the cells repopulate an organ. There is a need to show that the cells derived from introduced adult stem cells are contributing directly to tissue function, and to improve the ability to maintain adult stem cells in culture without the cells differentiating. Finally, most of the studies that have garnered so much attention have used mouse rather than human adult stem cells.
ES cells are not without their own potential problems as a source of cells for transplantation. The growth of human ES cells in culture requires a “feeder” layer of mouse cells that may contain viruses, and when allowed to differentiate the ES cells can form a mixture of cell types at once. Human ES cells can form benign tumors when introduced into mice , although this potential seems to disappear if the cells are allowed to differentiate before introduction into a recipient . Studies with mouse ES cells have shown promise for treating diabetes , Parkinson’s disease , and spinal cord injury .
The ES cells made with nuclear transplantation would have the advantage over adult stem cells of being able to provide virtually all cell types and of being able to be maintained in culture for long periods of time. Current knowledge is, however, uncertain, and research on both
adult stem cells and stem cells made with nuclear transplantation is required to understand their therapeutic potentials. (This point is stated clearly in Finding and Recommendation 2 of Stem Cells and the Future of Regenerative Medicine  which states, in part, that “studies of both embryonic and adult human stem cells will be required to most efficiently advance the scientific and therapeutic potential of regenerative medicine.”) It is likely that the ES cells will initially be used to generate single cell types for transplantation, such as nerve cells or muscle cells. In the future, because of their ability to give rise to many cell types, they might be used to generate tissues and, theoretically, complex organs for transplantation. But this will require the perfection of techniques for directing their specialization into each of the component cell types and then the assembly of these cells in the correct proportion and spatial organization for an organ. That might be reasonably straightforward for a simple structure, such as a pancreatic islet that produces insulin, but it is more challenging for tissues as complex as that from lung, kidney, or liver [54; 55].
The experimental procedures required to produce stem cells through nuclear transplantation would consist of the transfer of a somatic cell nucleus from a patient into an enucleated egg, the in vitro culture of the embryo to the blastocyst stage, and the derivation of a pluripotent ES cell line from the inner cell mass of this blastocyst. Such stem cell lines would then be used to derive specialized cells (and, if possible, tissues and organs) in laboratory culture for therapeutic transplantation. Such a procedure, if successful, can avoid a major cause of transplant rejection. However, there are several possible drawbacks to this proposal. Experiments with animal models suggest that the presence of divergent mitochondrial proteins in cells may create “minor” transplantation antigens [56; 57] that can cause rejection [58-63]; this would not be a problem if the egg were donated by the mother of the transplant recipient or the recipient herself. For some autoimmune diseases, transplantation of cells cloned from the patient’s own cells may be inappropriate, in that these cells can be targets for the ongoing destructive process. And, as with the use of adult stem cells, in the case of a disorder that has a genetic origin, ES cells derived by nuclear transplantation from the patient’s own cells would carry the same defect and would have to be grown and genetically modified before they could be used for therapeutic transplantation. Using another source of stem cells is more likely to be feasible (although immunosuppression would be required) than the challenging task of correcting the one or more genes that are involved in the disease in adult stem cells or in a nuclear transplantation-derived stem cell line initiated with a nucleus from the patient.
In addition to nuclear transplantation, there are two other methods by which researchers might be able to derive ES cells with reduced likeli-
hood for rejection. A bank of ES cell lines covering many possible genetic makeups is one possibility, although the National Academies report entitled Stem Cells and the Future of Regenerative Medicine rated this as “difficult to conceive” . Alternatively, embryonic stem cells might be engineered to eliminate or introduce certain cell-surface proteins, thus making the cells invisible to the recipient’s immune system. As with the proposed use of many types of adult stem cells in transplantation, neither of these approaches carries anything close to a promise of success at the moment.
The preparation of embryonic stem cells by nuclear transplantation differs from reproductive cloning in that nothing is implanted in a uterus. The issue of whether ES cells alone can give rise to a complete embryo can easily be misinterpreted. The titles of some reports suggest that mouse embryos can be derived from ES cells alone [64-72]. In all cases, however, the ES cells need to be surrounded by cells derived from a host embryo, in particular trophoblast and primitive endoderm. In addition to forming part of the placenta, trophoblast cells of the blastocyst provide essential patterning cues or signals to the embryo that are required to determine the orientation of its future head and rump (anterior-posterior) axis. This positional information is not genetically determined but is acquired by the trophoblast cells from events initiated soon after fertilization or egg activation. Moreover, it is critical that the positional cues be imparted to the inner cells of the blastocyst during a specific time window of development [73-76]. Isolated inner cell masses of mouse blastocysts do not implant by themselves, but will do so if combined with trophoblast vesicles from another embryo . By contrast, isolated clumps of mouse ES cells introduced into trophoblast vesicles never give rise to anything remotely resembling a postimplantation embryo, as opposed to a disorganized mass of trophoblast. In other words, the only way to get mouse ES cells to participate in normal development is to provide them with host embryonic cells, even if these cells do not remain viable throughout gestation (Richard Gardner, personal communication). It has been reported that human  and primate [78-79] ES cells can give rise to trophoblast cells in culture. However, these trophoblast cells would presumably lack the positional cues normally acquired during the development of a blastocyst from an egg. In the light of the experimental results with mouse ES cells described above, it is very unlikely that clumps of human ES cells placed in a uterus would implant and develop into a fetus. It has been reported that clumps of human ES cells in culture, like clumps of mouse ES cells, give rise to disorganized aggregates known as embryoid bodies .
Besides their uses for therapeutic transplantation, ES cells obtained by nuclear transplantation could be used in laboratories for several types of studies that are important for clinical medicine and for fundamental research in human developmental biology. Such studies could not be
carried out with mouse or monkey ES cells and are not likely to be feasible with ES cells prepared from normally fertilized blastocysts. For example, ES cells derived from humans with genetic diseases could be prepared through nuclear transplantation and would permit analysis of the role of the mutated genes in both cell and tissue development and in adult cells difficult to study otherwise, such as nerve cells of the brain. This work has the disadvantage that it would require the use of donor eggs. But for the study of many cell types there may be no alternative to the use of ES cells; for these cell types the derivation of primary cell lines from human tissues is not yet possible.
If the differentiation of ES cells into specialized cell types can be understood and controlled, the use of nuclear transplantation to obtain genetically defined human ES cell lines would allow the generation of genetically diverse cell lines that are not readily obtainable from embryos that have been frozen or that are in excess of clinical need in IVF clinics. The latter do not reflect the diversity of the general population and are skewed toward genomes from couples in which the female is older than the period of maximal fertility or one partner is infertile. In addition, it might be important to produce stem cells by nuclear transplantation from individuals who have diseases associated with both simple  and complex (multiple-gene) heritable genetic predilections. For example, some people have mutations that predispose them to “Lou Gehrig’s disease” (amyotrophic lateral sclerosis, or ALS); however, only some of these individuals become ill, presumably because of the influence of additional genes. Many common genetic predilections to diseases have similarly complex etiologies; it is likely that more such diseases will become apparent as the information generated by the Human Genome Project is applied. It would be possible, by using ES cells prepared with nuclear transplantation from patients and healthy people, to compare the development of such cells and to study the fundamental processes that modulate predilections to diseases.
Neither the work with ES cells, nor the work leading to the formation of cells and tissues for transplantation, involves the placement of blastocysts in a uterus. Thus, there is no embryonic development beyond the 64 to 200 cell stage, and no fetal development.
2-1. Reproductive cloning involves the creation of individuals that contain identical sets of nuclear genetic material (DNA). To have complete genetic identity, clones must have not only the same nuclear genes, but also the same mitochondrial genes.
2-2. Cloned mammalian animals can be made by replacing the chromosomes of an egg cell with a nucleus from the individual to be cloned, followed by stimulation of cell division and implantation of the resulting embryo.
2-3. Cloned individuals, whether born at the same or different times, will not be physically or behaviorally identical with each other at comparable ages.
2-4. Stem cells are cells that have an extensive ability to self-renew and differentiate, and they are therefore important as a potential source of cells for therapeutic transplantation. Embryonic stem cells derived through nuclear transplantation into eggs are a potential source of pluripotent (embryonic) stem cell lines that are immunologically similar to a patient’s cells. Research with such cells has the goal of producing cells and tissues for therapeutic transplantation with minimal chance of rejection.
2-5. Embryonic stem cells and cell lines derived through nuclear transplantation could be valuable for uses other than organ transplantation. Such cell lines could be used to study the heritable genetic components associated with predilections to a variety of complex genetic diseases and test therapies for such diseases when they affect cells that are hard to study in isolation in adults.
2-6. The process of obtaining embryonic stem cells through nuclear transplantation does not involve the placement of an embryo in a uterus, and it cannot produce a new individual.
1. COLMAN A. Somatic cell nuclear transfer in mammals: Progress and applications. Cloning 1999, 1(4):185-200.
2. WOLF E, ZAKHARTCHENKO V, BREM G. Nuclear transfer in mammals: recent developments and future perspectives. J Biotechnol 1998 Oct 27(65) 2-3:99-110.
3. CHAN AW, DOMINKO T, LUETJENS CM, NEUBER E, MARTINOVICH C, HEWITSON L, SIMERLY CR, SCHATTEN GP. Clonal propagation of primate offspring by embryo splitting. Science 2000 Jan 14, 287(5451):317-319.
4. HALL JG. Twinning: mechanisms and genetic implications. Curr Opin Genet Dev 1996 Jun, 6(3):343-7.
5. HALL JG. Genomic imprinting: nature and clinical relevance. Annu Rev Med 1997, 48:35-44.
6. SIMON DK, JOHNS DR. Mitochondrial disorders: clinical and genetic features. Annu Rev Med 1999, 50:111-27.
7. FINNILA S, AUTERE J, LEHTOVIRTA M, HARTIKAINEN P, MANNERMAA A, SOININEN H, MAJAMAA K. Increased risk of sensorineural hearing loss and migraine in patients with a rare mitochondrial DNA variant 4336A>G in tRNAGln. J Med Genet 2001 Jun, 38(6):400-5.
8. MCCREATH KJ, HOWCROFT J, CAMPBELL KH, COLMAN A, SCHNIEKE AE, KIND AJ. Production of gene-targeted sheep by nuclear transfer from cultured somatic cells. Nature 2000 Jun 29, 405(6790):1066-9.
9. SCHNIEKE AE, KIND AJ, RITCHIE WA, MYCOCK K, SCOTT AR, RITCHIE M, WILMUT I, COLMAN A, CAMPBELL KH. Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts. Science 1997 Dec 19, 278(5346):2130-3.
10. FIDDLER M, PERGAMENT D, PERGAMENT E. The role of the preimplantation geneticist in human cloning. Prenat Diagn 1999 Dec, 19(13):1200-4.
11. COMMITTEE ON STEM CELLS AND THE FUTURE OF REGENERATIVE MEDICINE, BOARD ON LIFE SCIENCES AND BOARD ON NEUROSCIENCE AND BEHAVIORAL HEALTH. Stem Cells and the Future of Regenerative Medicine. Report of the National Academy of Sciences and the Institute of Medicine. 2001 Sep.
12. BAUM CM, WEISSMAN IL, TSUKAMOTO AS, BUCKLE AM, PEAULT B. Isolation of a candidate human hematopoietic stem-cell population. Proc Natl Acad Sci U S A 1992 Apr 01, 89(7):2804-8.
13. AZIZI SA, STOKES D, AUGELLI BJ, DIGIROLAMO C, PROCKOP DJ. Engraftment and migration of human bone marrow stromal cells implanted in the brains of albino rats—similarities to astrocyte grafts. Proc Natl Acad Sci U S A 1998 Mar 31, 95(7):3908-13.
14. UCHIDA N, BUCK DW, HE D, REITSMA MJ, MASEK M, PHAN TV, TSUKAMOTO AS, GAGE FH, WEISSMAN IL. Direct isolation of human central nervous system stem cells. Proc Natl Acad Sci U S A 2000 Dec 19, 97(26):14720-5.
15. PALMER TD, SCHWARTZ PH, TAUPIN P, KASPAR B, STEIN SA, GAGE FH. Cell culture. Progenitor cells from human brain after death. Nature 2001 May 03, 411(6833):42-3.
16. ZUK PA, ZHU M, MIZUNO H, HUANG J, FUTRELL JW, KATZ AJ, BENHAIM P, LORENZ HP, HEDRICK MH. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 2001 Apr, 7(2):211-28.
17. KRAUSE DS, THEISE ND, COLLECTOR MI, HENEGARIU O, HWANG S, GARDNER R, NEUTZEL S, SHARKIS SJ. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell 2001 May 04, 105(3):369-77.
18. TOMA JG, AKHAVAN M, FERNANDES KJL, BARNABÉ-HEIDER F, SADIKOT A, KAPLAN DR, MILLER FD. Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nature Cell Biology 2001 Sep, 3 778-784.
19. RIETZE RL, VALCANIS H, BROOKER GF, THOMAS T, VOSS AK, BARTLETT PF. Purification of a pluripotent neural stem cell from the adult mouse brain. Nature 2001 Aug 16, 412(6848):736-9.
20. THOMSON JA, ITSKOVITZ-ELDOR J, SHAPIRO SS, WAKNITZ MA, SWIERGIEL JJ, MARSHALL VS, JONES JM. Embryonic stem cell lines derived from human blastocysts. Science 1998 Nov 06, 282(5391):1145-7.
21. EVANS MJ, KAUFMAN MH. Establishment in culture of pluripotential cells from mouse embryos. Nature 1981 Jul 09, 292(5819):154-6.
22. MARTIN GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A 1981 Dec, 78(12):7634-8.
23. REUBINOFF BE, PERA MF, FONG CY, TROUNSON A, BONGSO A. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 2000 Apr, 18(4):399-404.
24. SHINOHARA T, BRINSTER RL. Enrichment and transplantation of spermatogonial stem cells. Int J Androl 2000, 23 Suppl 2:89-91.
25. WEISSMAN IL. Translating stem and progenitor cell biology to the clinic: Barriers and opportunities. Science 2000 Feb 25, 287(5457):1442-6.
26. LAGASSE E, SHIZURU JA, UCHIDA N, TSUKAMOTO A, WEISSMAN IL. Toward regenerative medicine. Immunity 2001 Apr, 14(4):425-36.
27. GUSSONI E, SONEOKA Y, STRICKLAND CD, BUZNEY EA, KHAN MK, FLINT AF, KUNKEL LM, MULLIGAN RC. Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature 1999 Sep 23, 401(6751):390-4.
28. LEE SH, LUMELSKY N, STUDER L, AUERBACH JM, MCKAY RD. Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells. Nat Biotechnol 2000 Jun, 18(6):675-9.
29. WAKAYAMA T, TABAR V, RODRIGUEZ I, PERRY AC, STUDER L, MOMBAERTS P. Differentiation of embryonic stem cell lines generated from adult somatic cells by nuclear transfer. Science 2001 Apr 27, 292(5517):740-3.
30. LUMELSKY N, BLONDEL O, LAENG P, VELASCO I, RAVIN R, MCKAY R. Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Science 2001 May 18, 292(5520):1389-94.
31. SHAMBLOTT MJ, AXELMAN J, LITTLEFIELD JW, BLUMENTHAL PD, HUGGINS GR, CUI Y, CHENG L, GEARHART JD. Human embryonic germ cell derivatives express a broad range of developmentally distinct markers and proliferate extensively in vitro. Proc Natl Acad Sci U S A 2001 Jan 02, 98(1):113-8.
32. NEGRIN RS, ATKINSON K, LEEMHUIS T, HANANIA E, JUTTNER C, TIERNEY K, HU WW, JOHNSTON LJ, SHIZURN JA, STOCKERL-GOLDSTEIN KE, BLUME KG, WEISSMAN IL, BOWER S, BAYNES R, DANSEY R, KARANES C, PETERS W, KLEIN J. Transplantation of highly purified CD34+Thy-1+ hematopoietic stem cells in patients with metastatic breast cancer. Biol Blood Marrow Transplant 2000, 6(3):262-71.
33. FERRARI G, CUSELLA-DE ANGELIS G, COLETTA M, PAOLUCCI E, STORNAIUOLO A, COSSU G, MAVILIO F. Muscle regeneration by bone marrow-derived myogenic progenitors. Science 1998 Mar 06, 279(5356):1528-30.
34. PETERSEN BE, BOWEN WC, PATRENE KD, MARS WM, SULLIVAN AK, MURASE N, BOGGS SS, GREENBERGER JS, GOFF JP. Bone marrow as a potential source of hepatic oval cells. Science 1999 May 14, 284(5417):1168-70.
35. ALISON MR, POULSOM R, JEFFERY R, DHILLON AP, QUAGLIA A, JACOB J, NOVELLI M, PRENTICE G, WILLIAMSON J, WRIGHT NA. Hepatocytes from non-hepatic adult stem cells. Nature 2000 Jul 20, 406(6793):257.
36. BONNER-WEIR S, TANEJA M, WEIR GC, TATARKIEWICZ K, SONG KH, SHARMA A, O’NEIL JJ. In vitro cultivation of human islets from expanded ductal tissue. Proc Natl Acad Sci U S A 2000 Jul 05, 97(14):7999-8004.
37. CLARKE DL, JOHANSSON CB, WILBERTZ J, VERESS B, NILSSON E, KARLSTROM H, LENDAHL U, FRISEN J. Generalized potential of adult neural stem cells. Science 2000 Jun 02, 288(5471):1660-3.
38. LAGASSE E, CONNORS H, AL-DHALIMY M, REITSMA M, DOHSE M, OSBORNE L, WANG X, FINEGOLD M, WEISSMAN IL, GROMPE M. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med 2000 Nov, 6(11):1229-34.
39. MEZEY E, CHANDROSS KJ, HARTA G, MAKI RA, MCKERCHER SR. Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science 2000 Dec 01, 290(5497):1779-82.
40. FALLON J, REID S, KINYAMU R, OPOLE I, OPOLE R, BARATTA J, KORC M, ENDO TL, DUONG A, NGUYEN G, KARKEHABADHI M, TWARDZIK D, PATEL S, LOUGHLIN S. In vivo induction of massive proliferation, directed migration, and differentiation of neural cells in the adult mammalian brain. Proc Natl Acad Sci U S A 2000 Dec 19, 97(26):14686-91.
41. BRAZELTON TR, ROSSI FM, KESHET GI, BLAU HM. From marrow to brain: expression of neuronal phenotypes in adult mice. Science 2000 Dec 01, 290(5497):1775-9.
42. KOCHER AA, SCHUSTER MD, SZABOLCS MJ, TAKUMA S, BURKHOFF D, WANG J, HOMMA S, EDWARDS NM, ITESCU S. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med 2001 Apr, 7(4):430-6.
43. ANDERSON DJ, GAGE FH, WEISSMAN IL. Can stem cells cross lineage boundaries? Nat Med 2001 Apr, 7(4):393-5.
44. LANZA RP, CAPLAN AL, SILVER LM, CIBELLI JB, WEST MD, GREEN RM. The ethical validity of using nuclear transfer in human transplantation. JAMA 2000 Dec 27, 284(24):3175-9.
45. WEISSMAN IL, BALTIMORE D. Disappearing stem cells, disappearing science. Science 2001 Apr 27, 292(5517):601.
46. WINSTON R. Embryonic stem cell research: The case for… Nat Med 2001 Apr, 7(4):396-397.
47. VOGEL G. Stem cell policy. Can adult stem cells suffice? Science 2001 Jun 08, 292(5523):1820-2.
48. GURDON JB, COLMAN A. The future of cloning. Nature 1999 Dec 16, 402(6763):743-6.
49. PERA MF, REUBINOFF B, TROUNSON A. Human embryonic stem cells. J Cell Sci 2000 Jan, 113(Pt 1):5-10.
50. ODORICO JS, KAUFMAN DS, THOMSON JA. Multilineage differentiation from human embryonic stem cell lines. Stem Cells 2001, 19(3):193-204.
51. STUDER L, TABAR V, MCKAY RD. Transplantation of expanded mesencephalic precursors leads to recovery in parkinsonian rats. Nat Neurosci 1998 Aug, 1(4):290-5.
52. MCDONALD JW, LIU XZ, QU Y, LIU S, MICKEY SK, TURETSKY D, GOTTLIEB DI, CHOI DW. Transplanted embryonic stem cells survive, differentiate and promote recovery in injured rat spinal cord. Nat Med 1999 Dec, 5(12):1410-2.
53. LANZA RP, CIBELLI JB, WEST MD. Prospects for the use of nuclear transfer in human transplantation. Nat Biotechnol 1999 Dec, 17(12):1171-4.
54. MUNSIE MJ, MICHALSKA AE, O’BRIEN CM, TROUNSON AO, PERA MF, MOUNTFORD PS. Isolation of pluripotent embryonic stem cells from reprogrammed adult mouse somatic cell nuclei. Curr Biol 2000 Aug 24, 10(16):989-92.
55. SIMPSON E. Minor transplantation antigens: animal models for human host-versus-graft, graft-versus-host, and graft-versus-leukemia reactions. Transplantation 1998 Mar 15, 65(5):611-6.
56. SIMPSON E, ROOPENIAN D. Minor histocompatibility antigens. Curr Opin Immunol 1997 Oct, 9(5):655-61.
57. CHAN T, FISCHER LINDAHL K. Skin graft rejection caused by the maternally transmitted antigen Mta. Transplantation 1985 May, 39(5):477-80.
58. FISCHER LINDAHL K, HERMEL E, LOVELAND BE, WANG CR. Maternally transmitted antigen of mice: a model transplantation antigen. Annu Rev Immunol 1991, 9:351-72.
59. DAVIES JD, SILVERS WK, WILSON DB. A transplantation antigen, possibly of mitochondrial origin, that elicits rejection of parental strain skin grafts by F1 rats. Transplantation 1992 Oct, 54(4):730-1.
60. DABHI VM, LINDAHL KF. MtDNA-encoded histocompatibility antigens. Methods Enzymol 1995, 260:466-85.
61. DABHI VM, LINDAHL KF. CTL respond to a mitochondrial antigen presented by H2-Db. Immunogenetics 1996, 45(1):65-8.
62. BHUYAN PK, YOUNG LL, LINDAHL KF, BUTCHER GW. Identification of the rat maternally transmitted minor histocompatibility antigen. J Immunol 1997 Apr 15, 158(8):3753-60.
63. AMANO T, KATO Y, TSUNODA Y. Comparison of heat-treated and tetraploid blastocysts for the production of completely ES-cell-derived mice. Zygote 2001 May, 9(2):153-7.
64. AMANO T, NAKAMURA K, TANI T, KATO Y, TSUNODA Y. Production of mice derived entirely from embryonic stem cells after injecting the cells into heat treated blastocysts. Theriogenology 2000 Apr 15, 53(7):1449-58.
65. EGGAN K, AKUTSU H, LORING J, JACKSON-GRUSBY L, KLEMM M, RIDEOUT WM 3rd, YANAGIMACHI R, JAENISCH R. Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation. Proc Natl Acad Sci U S A 2001 May 22, 98(11):6209-14.
66. IWASAKI S, CAMPBELL KH, GALLI C, AKIYAMA K. Production of live calves derived from embryonic stem-like cells aggregated with tetraploid embryos. Biol Reprod 2000 Feb, 62(2):470-5.
67. NAGY A, GOCZA E, DIAZ EM, PRIDEAUX VR, IVANYI E, MARKKULA M, ROSSANT J. Embryonic stem cells alone are able to support fetal development in the mouse. Development 1990 Nov, 110(3):815-21.
68. NAGY A, ROSSANT J, NAGY R, ABRAMOW-NEWERLY W, RODER JC. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc Natl Acad Sci U S A 1993 Sep 15, 90(18):8424-8.
69. TANAKA M, HADJANTONAKIS AK, NAGY A. Aggregation chimeras. Combining ES cells, diploid and tetraploid embryos . Methods Mol Biol 2001, 158:135-54.
70. UEDA O, JISHAGE K, KAMADA N, UCHIDA S, SUZUKI H. Production of mice entirely derived from embryonic stem (ES) cell with many passages by coculture of ES cells with cytochalasin B induced tetraploid embryos. Exp Anim 1995 Jul, 44(3):205-10.
71. WANG ZQ, KIEFER F, URBANEK P, WAGNER EF. Generation of completely embryonic stem cell-derived mutant mice using tetraploid blastocyst injection. Mech Dev 1997 Mar, 62(2):137-45.
72. BEDDINGTON RS, ROBERTSON EJ. Axis development and early asymmetry in mammals. Cell 1999 Jan 22, 96(2):195-209.
73. GARDNER RL. Axial relationships between egg and embryo in the mouse. Curr Top Dev Biol 1998, 39:35-71.
74. GARDNER RL. The initial phase of embryonic patterning in mammals. Int Rev Cytol 2001, 203:233-90.
75. GARDNER RL. Specification of embryonic axes begins before cleavage in normal mouse development. Development 2001 Mar, 128(6):839-47.
76. GARDNER, R. L. An investigation of inner cell mass and trophoblast tissues following their isolation from the mouse blastocyst. J. Embryol exp. Morphology 1972(28):279-312.
77. THOMSON JA, MARSHALL VS. Primate embryonic stem cells. Curr Top Dev Biol 1998, 38:133-65.
78. THOMSON JA, KALISHMAN J, GOLOS TG, DURNING M, HARRIS CP, BECKER RA, HEARN JP. Isolation of a primate embryonic stem cell line. Proc Natl Acad Sci USA 1995 Aug 15, 92(17):7844-8.
79. ITSKOVITZ-ELDOR, J., SCHULDINER, M., KARSENTI, D., EDEN, A., YANUKA, O., AMIT, M., SOREQ, H., AND BENVENISTY, N. Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol Med. 2000(6):88-95.
80. RECHITSKY S, STROM C, VERLINSKY O, AMET T, IVAKHNENKO V, KUKHARENKO V, KULIEV A, VERLINSKY Y. Accuracy of preimplantation diagnosis of single-gene disorders by polar body analysis of oocytes. J Assist Reprod Genet 1999 Apr, 16(4):192-8.