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A new era was heralded by the birth of Dolly, born in July 1996 after transfer of the nucleus from a mammary cell of an adult ewe to an enucleated oocyte. In this editorial review, we describe the research that led to the birth of Dolly and discuss some of the implications of the Dolly experiment in changing the way we think about cell plasticity.
Research That Led to the Birth of Dolly
Dolly was the first animal to be born following transfer of a nucleus from an adult donor. Although this success followed research over several decades in amphibians and laboratory mammals, it followed specifically from the recognition of the need to co-ordinate the cell cycles of donor nucleus and recipient oocyte. There had been recognition of the importance of this relationship, but the work of Keith Campbell at Roslin provided the first detailed description of the changes in level of regulatory molecules (meiosis/mitosis promoting factor) in the mammalian oocyte and the early embryo after fertilization, and the effect on transferred nuclei.
This research suggested two protocols for nuclear transfer in sheep that are expected to produce embryos with normal ploidy. In one, the oocyte is activated before enucleation and transfer of a nucleus, which may be at any stage of the cycle. In this case, the transferred nucleus determines whether or not DNA replication occurs. In the second protocol, nuclei that are awaiting DNA replication are transferred into oocytes in metaphase II of meiosis. In this case, DNA replication occurs in an appropriate manner because the transferred nucleus is awaiting replication and the cytoplast is primed to carry out the replication. All other combinations are expected to lead to errors of one kind or another and have to have limited developmental potential.
Optimization of the Roslin Procedures
This initial analysis defined procedures that yield embryos with normal ploidy. Further research demonstrated that a greater proportion of reconstructed embryos developed to term if they were produced by transfer of a nucleus in G0/G1 to an oocyte in metaphase II. This difference is thought to reflect the greater efficiency with which the oocyte cytoplasm reprograms gene expression of the transferred somatic cell nucleus to that of an early embryo.
Production of embryonic cells in G1-phase is time-consuming and not entirely accurate. This is particularly the case for embryo-derived cells, which do not respond to the normal checkpoints. Keith Campbell introduced the use of cultured cells in G0, through which tissue-derived cells exit the cell cycle and become quiescent if cultured in a low concentration of serum. In this case, the quiescent cells are in a stable state and can be left in an incubator or on the bench for as long as nuclei are required for transfer. This innovation facilitated nuclear transfer and yielded more reproducible results.
Modifications to the Roslin Protocol
These analyses led to the birth of Dolly and provided the biological basis for research in other species. In most species, viable offspring were obtained using the Roslin procedure. Primates and rats were two exceptions.
When the Roslin procedure was applied in human and nonhuman primates, development was limited to the blastocyst stage until it was discovered that handling and enucleation of primate oocytes according to existing procedures induced partial activation of the oocyte. It was noted that under these circumstances, the activation procedure applied after nuclear transfer was unable to induce a full response in the oocyte and development of reconstructed embryos was limited.
Procedures for handling and enucleation had been modified so as not to induce premature activation. It is argued by some practitioners that human embryo stem cell lines from cloned embryos offer the best possible cells for use in cell therapy. In particular, this is because they would be genetically identical to the patient, if the nuclear donor cell is taken from the patient. This approach contrasts with that of using iPS cells that are homozygous at major HLA loci to provide useful partial match to recipients.
Progress in the rat was hindered by technical limitations such as poor-quality culture systems that failed to culture embryos beyond a two- to four-cell block, and problems with spontaneous activation of oocytes on removal from the oviduct. These issues were overcome through modifications to the culture medium and the use of a proteasome inhibitor (MG132) to stabilize the oocytes at metaphase II. Somatic nuclear transfer in the rat remains problematic, since the publication in 2003 describing the birth of two viable offspring using the Honolulu mouse protocol, subsequent publications have failed to report births of live cloned rat pups.
Precise Genetic Modification of Livestock
The cloning project at Roslin Institute was initiated with the aim of being able to introduce precise genetic modification into livestock. This would be achieved by introduction of the desired change into nuclei from a donor with a suitable background. After confirmation of the desired genetic change, these cells would be used for nuclear transfer. The resulting offspring would have the characteristics of the selected animal with the additional, selected modification. As a demonstration, the group at Roslin used this approach to introduce sequences in which regulatory sequences from CASEIN/BETA lactoglobulin directed production of human clotting factor IX into the milk of sheep.
The Roslin procedure for nuclear transfer has been used by others to introduce genetic change, for example, to create models of the human genetic disease cystic fibrosis in pigs. While this protocol was effective, a more efficient alternative procedure for the introduction of precise genetic changes has been provided by the development of the “clustered, regularly interspaced, short palindromic repeat” (CRISPR) technology described by Sander and Joung. These systems are so efficient and accurate that it is apparently possible to simultaneously modify several genes in mouse embryos using the CRISPR-Cas 9 system, and with optimization this may also be possible in livestock species.
Implications of the Dolly Experiment
The birth of viable offspring following transfer of nuclei from cells of adult donors demonstrated that differentiation does not depend on loss of chromosomal DNA from the donor cells during the course of development, as had been suggested by Weissman et al. in 1888. More striking was the observation that differentiation of the nucleus could be reversed. This result contrasted with the outcome following previous attempts to dedifferentiate adult somatic cells.
The birth of Dolly stimulated a number of groups to consider if they could find other ways of changing cells from one phenotype to another, most frequently to obtain pluripotent cells from populations of somatic cells. If unknown factors in the recipient oocyte could “reprogram” the nucleus to a stage very early in development, then there might be other ways of making that change. Within 10 years, two laboratories (Yamanaka and Thomson) working independently established very similar protocols by which the introduction of selected transcription factors changes a small proportion of the treated cells to pluripotent cells. These “induced pluripotent stem cells” (iPS cells) were very similar indeed to human embryo-derived pluripotent stem cells (hES cells). These cells were obtained following the introduction of a small number of selected transcription factors into somatic cells such as skin fibroblasts or peripheral blood cells. The factors are known either to be essential for normal function of ES cells or are only expressed in ES cells.
It is now possible to produce iPS cells in chemically defined media suitable for derivation of cells for clinical use. The ability to produce iPS cells is providing revolutionary new opportunities in research and cell therapy. A considerable research effort is directed now to the establishment of procedures for the derivation of iPS cells at clinical grade and their differentiation to tissue types that are required for cell therapy. Small trials are beginning with some encouraging results. However, having in mind the requirement for detailed clinical trials, it will be several decades before cell therapy becomes possible on a large scale.
Studies of Degenerative Diseases
In addition, iPS cell lines are being derived from donors who have a degenerative disease. In some cases, cells are donated by patients who have an inherited disease. iPS cells from these donors provide the first opportunity to study the development of the disease and to compare normal and diseased development. These studies may provide the opportunity to devise assays that can be used to search for small molecules to reduce the harmful effect of the causative mutation. Effective small molecules would then be assessed in animal models before clinical trials are considered. Around the world research is in progress with several different diseases, including Amyotrophic lateral sclerosis (ALS), Parkinson's disease, blindness, and heart disease.
It seems likely that if iPS cells can be differentiated to the cells that are damaged in the disease in question, then this approach could be used to search for small molecules able to provide treatment for many degenerative diseases, for which there is no treatment at present.
Cell Therapy
Biologists have always been attracted by the possibility of being able to replace cells that have either died or ceased to function normally, leading to degenerative diseases such as ALS, Parkinson's disease, type 1 diabetes, spinal cord injury, some forms of blindness, or heart failure. There are a number of practical difficulties yet to be overcome. The scale of production that would be required to meet this need is enormous, while maintaining a consistent high standard of both cleanliness and of appropriate tissue-specific character. Finally, there will be the need to prevent immune rejection of transplanted cells.
A group led by Craig Taylor of Addenbrookes Hospital Cambridge has considered the position and put forward some conclusions and suggestions. First, that cells derived from human iPS cells would soon be of a suitable standard for transplantation. Second, that it would be impracticable to consider large scale use of patient-specific lines. By contrast, Craig and others have considered the use of donors who are homozygous at major HLA antigens. Typically, such cells offer a useful partial match to a larger number of recipients. A detailed analysis of data from the U.K. population predicted that a library of just over 150 lines selected according to this strategy would provide a useful partial match to over 90% of the U.K. population. The number of cells required for transplantation would not be affected by use of this strategy, but the number of lines could be dramatically reduced.
It is clearly essential that the effectiveness of this strategy is assessed thoroughly as quickly as possible, but experience gained from organ transplantation lends great hope that it will be effective, and so renders large-scale use of cell therapy a realistic possibility.