Nuclear transplantation

A monoclonal mouse?

One potential use of nuclear-transplantation — cloning — technology is to generate genetically matched tissues for treating adult patients. But there's a debate about whether mature adult cells are a good source of nuclei.

Behind all the fuss about the cloning of animals and prospects for cloning humans, some interesting scientific issues remain. For instance, can the nucleus of even the most specialized adult cell be completely 'reprogrammed', so that it can direct the development of a whole new embryo? Or are there subsets of cells in adult organisms — perhaps representing the unspecialized cells known as stem cells — that can be reset more easily? Hochedlinger and Jaenisch1 tackle the question on page 1035 of this issue, and claim success in producing cloned mice by using nuclei from mature T or B cells. The results provide strong evidence that the nucleus of a differentiated cell can indeed be reprogrammed.

Given all the current hype, it might seem odd that the most commonly used approach to cloning — 'somatic-cell nuclear transfer' — was in fact developed some 40 years ago in amphibians purely to address such fundamental questions. The technique involves replacing the nucleus of an egg with that of a more specialized cell from an embryo or adult. The idea is that the egg's cytoplasm somehow reprogrammes the nucleus so that it is as developmentally versatile as that of a fertilized egg, and able to direct the formation of all the tissues that make up an organism — which would, of course, be genetically identical to the original cell.

These early experiments showed that nuclei from morphologically differentiated amphibian cells, such as tadpole intestinal epithelial cells2 or adult skin cells3, could generate cloned tadpoles. But the success rate was low, and no adult frog was ever produced from an adult cell nucleus. By contrast, more recent experiments in mammals have shown that adult cell nuclei can produce viable clones4,5. But so few live-born offspring were obtained that it was possible that they did not come from mature cells, but from rare, less specialized adult cells such as stem cells.

So can highly differentiated adult cells be reprogrammed? To find out, some heritable mark is required that can unambiguously identify clones derived from a given differentiated cell. In mammals, the lymphocytes of the immune system are ideal for such an analysis. B lymphocytes are cells that produce soluble antibody molecules (immunoglobulins) in response to antigens (such as viruses or bacteria). Immunoglobulins are made up of a variable region, which recognizes antigens, and a constant region that triggers immune responses. The genome encodes enough variable regions to respond to every type of antigen. But each mature B cell produces only one type of antibody, enabling it to respond to one antigen. To achieve this 'monoclonality', the cell rearranges its DNA to weld the variable region of choice with the constant region. Similarly, T lymphocytes respond to antigens through their antigen-receptor complex; this is also rearranged at the genomic level, so that each T cell expresses one type of receptor.

T and B cells are, therefore, rare examples of cells in which the genome sequence is altered as they mature. So if you use the DNA from a T cell or B cell for cloning by nuclear transfer, the genomic rearrangement should be detected in all the cells of the clones.

Unfortunately, lymphocyte nuclei seem to be rather recalcitrant to nuclear reprogramming. In Hochedlinger and Jaenisch's study1, the likelihood of a cloned mouse embryo reaching even the blastocyst stage — a very early point in development — was only 4%. To get around this, the authors generated embryonic stem (ES) cells from the cloned blastocysts, and used a technique called tetraploid complementation6 to produce mice from the ES-cell lines (Fig. 1). They made two such lines: 'LN1' had immunoglobulin gene rearrangements, whereas 'LN2' showed rearrangement of the T-cell antigen-receptor gene. So LN1 was derived from a B-lymphocyte nucleus and LN2 from a T-cell nucleus. From line LN1, Hochedlinger and Jaenisch generated viable mice that had rearranged immunoglobulin genes in all tissues. No live mice resulted from line LN2, although one dead fetus was delivered at term and a separate experiment showed that ES cells from line LN2 could contribute to several tissues, including the immune system, when combined with wild-type cells in chimaeras.

Figure 1: Hochedlinger and Jaenisch's technique1 for generating cloned mice, using nuclei from mature immune cells.
figure1

a, Lymphocytes were isolated from the lymph nodes, which consist almost entirely of mature B and T lymphocytes. These cells show rearrangements of immunoglobulin or T-cell antigen-receptor genes, respectively, whereas other tissues in the body have unrearranged genes. b, Nuclei were extracted from B or T cells and c, transferred into enucleated mouse eggs and cultured to the blastocyst stage of development. d, Embryonic stem (ES) cells were then generated from the cloned blastocysts and aggregated with tetraploid embryos (e), generated by electrofusion of two-cell embryos. This produces chimaeras in which the entire fetus is derived from ES cells but the placental structures are provided by the tetraploid cells. Resulting offspring are entirely ES-cell-derived and, if developed from successful transfer of a B- or T-lymphocyte nucleus, will show the appropriate gene rearrangements in all tissues.

So does this experiment settle the question of whether nuclei from fully differentiated adult cells can be reprogrammed to allow normal development? The evidence is compelling, although the purists will say no. Using the ES-cell intermediates may have allowed time for extra reprogramming. Moreover, use of the tetraploid-complementation approach meant that the authors could not assess whether correct nuclear reprogramming could occur in placental tissues (see Fig. 1). Given that placental problems are one of the most consistent defects in cloned mammals, this is a valid concern. Finally, the number of live cloned offspring produced per number of nuclear transfers was pitifully small — approaching one in a thousand. So we are still left wondering whether the nuclei of differentiated cells can only be reprogrammed under exceptional circumstances.

Why do nuclei from different adult tissues vary in the efficiency with which they can be reprogrammed? Could it be due to whether or not the tissues contain stem cells? That remains to be seen. The idea that stem-cell nuclei may be more easily reprogrammed came from using ES cells — which are even more developmentally versatile than adult stem cells — as a source of nuclei for nuclear transfer7,8. These studies showed that more cloned blastocysts developed to the point of live birth when the source of nuclei was ES cells rather than adult cells.

But fewer embryos produced from ES-cell nuclei reached the blastocyst stage, meaning that the overall percentages of live offspring per nuclear transfer were not very different. One problem with ES-cell-derived clones is that their expression of 'imprinted' genes — genes that are specifically expressed from either the maternally derived or the paternally derived chromosome, but not from both — is often abnormal. By contrast, a study9 of cloned mice derived from adult cell nuclei showed that several imprinted genes were expressed normally. So stem cells from fetal or adult tissues (see, for example, refs 10, 11) might be a better choice than ES cells for testing the reprogramming potential of stem-cell nuclei.

These questions are of fundamental interest, but of course have practical implications. One of the potential uses of cloning is in treating human diseases; the idea is to use some of a patient's nuclei to produce genetically identical early embryos, from which ES cells would be generated and used to grow healthy replacement tissues in vitro. But unless there is a real breakthrough in finding a source of adult nuclei that can be efficiently reprogrammed, all the talk about this 'therapeutic cloning' will come to nothing. In the largest study in mice to date12, only 35 ES-cell lines were generated from over a thousand nuclear transfers (an efficiency of just 3.4%). This will not be acceptable in humans, where eggs will be hard to come by. Reprogramming adult cells directly, without an oocyte intermediate, would seem a more viable alternative. Surely the time has come for the cloners to turn their attention to the molecular mechanisms of nuclear reprogramming in the egg, and to use the information to enhance the potential of adult cells for use in cell-based therapies.

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Correspondence to Janet Rossant.

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Rossant, J. A monoclonal mouse?. Nature 415, 967–969 (2002). https://doi.org/10.1038/415967a

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