J Am Soc Nephrol 15: 1140–1144, 2004 Somatic Cell Cloning: The Ultimate Form of Nuclear Reprogramming? JORGE A. PIEDRAHITA,* BASHIR MIR,* SCOTT DINDOT,* and SHAWN WALKER† *Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh; and †Department of Veterinary Anatomy and Public Health, College of Veterinary Medicine, Texas A&M University, College Station. Abstract. With the increasing difficulties associated with meet- with cloning and genetic modifications in somatic cells need to ing the required needs for organs used in transplantation, be continued to be developed and optimized. This includes not alternative approaches need to be considered. These include only an enhancement of the rate of homologous recombination the use of stem cells as potential sources of specialized cells, in somatic cells, but also a thorough understanding of the the ability to transdifferentiate cell types in culture, and the nuclear reprogramming process taking place during nuclear development of complete organs that can be used in humans. transfer. The understanding of this process is likely to have an All of the above goals will require a complete understanding of effect beyond the area of nuclear transfer and assist with better the factors affecting cell differentiation and nuclear reprogram- methods for transdifferentiation of mammalian cells. ming. To make this a reality, however, techniques associated The ever-expanding gap between availability of organs and The goals of generating a complete organism by nuclear the number of patients awaiting an organ transplant is of great transfer of a nuclei into an oocyte, or reproductive cloning, has concern. In 2003, 14,880 transplantations were carried out been pursued for many years. By use of frog (Rana pipiens) between January and August, yet more than 83,000 patients eggs, Briggs and King (2) described the first nuclear transfer were in the organ waiting list (based on data from the Organ experiment. Briefly, ovulated MII eggs were enucleated with a Procurement and Transplantation Network as of November 1, handmade needle, and cells from a developing frog were lysed 2003). This increasing divergence between availability of or- and their nuclei injected into the egg. Over the next 4 to 11 d, gan donors and the increasing number of patients in waiting the majority of the cleaved blastulae developed into normal lists has caused researchers to look into alternative methods to postneurula and tadpole stages, demonstrating the ability of the fulfill these needs. Whereas for complete organ replacement, injected nuclei to at least partially complete normal develop- xenotransplantation with pig organs is the main alternative, for ment. These experiments were the first to demonstrate that the tissue repair, other alternatives exist. The most promising to genes, which served as the templates for RNA production and date is the use of embryo-derived and adult-derived stem cells which were responsible for the production of proteins for early (1). Cloning by nuclear transfer has a key role to play both by embryonic development, were not lost or irreversibly modified assisting in the generation of swine with complex genetic during differentiation, allowing for reprogramming of their fate manipulations, and in the generation of human stem cells. if placed in the proper environment. The development of Although ethical concerns need to be considered for the appli- techniques for microsurgery, microscopy, activation, enucle- cation of these technologies, such discussion is beyond the ation, and embryo culture and the process of nuclear transfer scope of this review. This review will focus on the develop- were modified for mammalian oocytes and blastomeres. Initial ment of the cloning technology, the basic concept in nuclear experiments attempted to introduce a somatic nucleus by Sen- reprogramming, and recent advances in both understanding the dai virus–mediated cell fusion or a cell nucleus by direct epigenetic process of nuclear reprogramming and in enhancing injection into rabbit eggs; however, at best, these eggs only the ability to undertake complex genetic manipulation in this underwent a few divisions (3,4). Development of the technol- species. ogy continued with partial successes along the way (5,6) until Tsunoda et al. (7), produced live mice from the transfer of nuclei from the one-, two-, four- and eight-cell stage. Willad- Correspondence to Dr. Jorge A. Piedrahita, Department of Molecular Biomed- sen (8) was the first to report successful nuclear transfer in ical Sciences, College of Veterinary Medicine, North Carolina State Univer- sity, Raleigh, NC, 27606. Phone: 919-515-7407; Fax: 919-513-7301; E-mail: nonmurine species and developed the framework for the meth- [email protected] ods in use today; his protocol was very similar to current 1046-6673/1505-1140 cloning techniques in that cytochalasin B is used for enucle- Journal of the American Society of Nephrology ation, the first polar body and the adjacent potion of the Copyright © 2004 by the American Society of Nephrology cytoplasm is removed, and the cell is fused to the cytoplasm. DOI: 10.1097/01.ASN.0000110183.87476.05 His report of cloned lambs was quickly followed by the pro- J Am Soc Nephrol 15: 1140–1144, 2004 Somatic Cell Cloning 1141 duction of the first cloned cow (9) and a cloned piglet (10). event to occur by limiting accessibility of the required nuclear However, in all cases, embryonic cells were used as nuclear factors. Embryonic cells and stem cells, in contrast, must be donors. The conceptual breakthrough came with the generation able to respond quickly to developmental signals and activate of Dolly, the cloned sheep (11). In this instance, the nuclear and deactivate specific regions of the genome. As a result, the donor was not an early embryonic cell but a fully differentiated chromatin structure is in a more open configuration and thus mammary gland cell. Dolly was the living proof that the nuclei more readily available for reprogramming. In mice, extensive of a fully differentiated cell still contains all of the information experimentation has demonstrated that embryonic stem cells required for he development of a full organism if placed in the are more easily reprogrammed than other cell types (25). proper environment or “reprogrammed” properly. After Dolly, Similarly, fully differentiated cells such as mature B lympho- sheep, cattle, goats, pigs, zebra fish, rabbits, cats, rats, and a cytes could not be fully reprogrammed by nuclear transfer mule have been added to the list of animals generated through alone (26). somatic cell nuclear transfer (12–17). However, generation of Additionally, multiple investigators have reported signifi- all species through cloning is hindered by low viable birth rates cant clonal differences derived from the same cell line. These and high incidences of abnormalities. In a recent article, a differences have been attributed to DNA changes occurring comprehensive analysis of the health profiles of cloned animals during culture (27). We feel this interpretation does not fully produced to date indicates that 64% of cattle, 40% of sheep, account for clonal differences because the cells remain karyo- and 93% of mice exhibit some form of abnormality (18). A typically normal. An alternative explanation is partial differ- large percentage of these animals die during gestation or entiation of specific subclones, accompanied by chromatin shortly after birth (19–22). Various abnormalities are mani- compaction, and the subsequent problem in reprogramming. fested in animals derived from nuclear transfer. In cattle they Although attempts at modifying chromatin organization before include large offspring syndrome, diabetes, pulmonary hyper- cloning are ongoing, results to date are inconclusive. However, tension, dilated cardiomyopathy, internal hemorrhaging umbil- because of the availability of chemicals such as 5-azacytidine ical artery, viral infection, dystocia, kidney problems, leg mal- and trichostatin A, both capable of remodeling chromatin formations, pneumonia, heart defects, liver fibrosis, (28,29), it is likely that eventually a protocol will be developed osteoporosis, joint defects, anemia, and placental abnormali- that will permit unpacking of the heterochromatic regions in ties. In sheep, abnormalities include large offspring syndrome, terminal differentiated cells before cloning. arthritis, and kidney, liver, and brain defects. Mice exhibit A more troubling drawback in nuclear reprogramming is the obesity, large offspring syndrome, enlarged placentas, umbil- differences associated with methylation patterns of donor cell ical hernias, respiratory failure, and failure to foster pups. nuclei compared with sperm and oocyte DNA. Before that can Goats and pigs exhibit relatively few abnormalities, but they be addressed, however, some understanding of DNA methyl- include bacterial infections of the lungs (goats) and abnormal ation and its complexity is required. Methylation at cytosine- teat numbers, cleft lips, and malformed limbs (pigs) (18,23). guanine nucleotides (CpG dinucleotides) exists in virtually all These abnormalities are likely associated with inaccurate nu- vertebrates, many invertebrates, and most plants (30). CpG clear reprogramming of the injected nuclei. But what exactly is dinucleotides are typically clustered in GC-rich regions, nuclear reprogramming, and what does it entail? termed CpG islands, but methylation