Factors affecting the developmental potential of cloned mammalian embryos.

F recent scientific advances have captured the imagination of biologists and the general public like the prospect of animal cloning (1, 2). The procedure is elegantly simple. A nucleus from a mature cell is transferred into the cytoplasm of an enucleated egg and becomes ‘‘reprogrammed’’ to re-execute embryogenesis. That cloning has been successful at all seems biologically remarkable and has forced biologists to assess what cell differentiation is all about. However, although possible, the process has many complications (3–5). Fetal and placental weight are often dramatically increased. Animals also frequently suffer from congenital anomalies and die within hours of birth. Embryonic and fetal losses are also extremely high, such that far less than 1% of manipulated embryos give rise to liveborn animals. These grim facts, collectively termed here cloned offspring syndrome, have raised considerable concern about the cloning process. The reasons for these complications have remained a mystery. However, work from the Jaenisch lab, culminating in a paper by Eggan et al. in this issue (6), has systematically addressed the problem and revealed surprising conclusions. Three potential causes of cloned offspring syndrome have been considered. As somatic cells are mostly used for cloning, it is argued that they have lost their developmental potential and can only rarely be reprogrammed (3, 7, 8). Surprisingly, this occurrence turns out not to be true. This was tested by using donor nuclei from embryonic stem (ES) cells. Murine ES cells, when injected into early embryos, contribute to all fetal structures, implying that they are totipotent. However, cloned embryos produced by using ES cell nuclei suffer a similar ill fate, in that only a tiny fraction of embryos survive to term and survive only rarely to adulthood (9, 10). Another popular idea is that culture of embryos prior to their transfer to the uterus introduces developmental errors (11). Studies with cattle and sheep embryos imply that indeed some fetal overgrowth results from procedures that involve embryo culture (11). However, the extent of this problem has not been directly compared with the effects of nuclear transfer. Notably, congenital anomalies and perinatal death are not associated with the culture of embryos. The final hypothesis is that the nuclear transfer process itself somehow results in complications of development. One of the problems in discriminating between these hypotheses is that all of the features of cloned offspring syndrome have been lumped together, an assumption that now appears to be incorrect. In earlier work with cloned mice made by transfer of ES cell nuclei, the Jaenisch lab made a fortuitous observation (9). Most ES cell lines are derived from inbred strains of mice. Although cloned mice derived from their 129ySv ES cell line invariably died perinatally, cloned animals derived from a (129y Sv 3 C57BLy6) F1 hybrid ES cell line survived to adulthood (9). In the present paper, they extended the observations to several other donor ES cell lines and found that embryonic development to term and postnatal survival were higher when F1 hybrid ES cells were used as the source of donor nuclei. Impressively, cloned animals survived to adulthood only when they were derived from F1 hybrid cells (Fig. 1) (6). Embryos completely derived from 129ySv ES cells, by making chimeras between the ES cells and host embryos, also develop to term at a low rate and typically die soon after birth (12). This finding raised the possibility that the poor developmental potential of cloned embryos derived from inbred strains was unrelated to the nuclear transfer process. To test this idea, embryos were generated by making ES cell-tetraploid chimeras (6). This approach takes advantage of the fact that blastomeres made tetraploid (4N) by electrofusion of a two-cell embryo replicate as tetraploid cells, which, although forming trophoblast and endoderm of the placenta and extraembryonic membranes, fail to form fetal structures. ES cells, by contrast, cannot form trophoblast and extraembryonic endoderm but do form fetal tissues. Chimeras made from ES cells and tetraploid embryos therefore form normal concepti because of complementary cell contributions (Fig. 1) (13). Strikingly, the fate of ES cell-derived fetuses produced by tetraploid complementation is similar to that of cloned embryos in that they survive to adulthood if they were derived from F1 hybrid ES cell lines (Fig. 1) (6). The results from the ES cell-tetraploid chimeras indicate that the poor postnatal survival of cloned mouse embryos is not because of the nuclear transfer process itself. But what about other aspects of cloned offspring syndrome? With respect to embryonic survival, F1 hybridderived embr yos develop to term at a significantly higher rate, whether they are produced as ES cell-tetraploid chimeras or via nuclear transfer. Notably, the rates of development from blastocyst to term were not different between the two methods (Fig. 1) (6). In contrast, one difference between embryos produced by cloning and tetraploid chimeras is that fetal, and particularly placental, size is significantly larger in the cloned animals. This is true for both inbred and F1 hybrid-derived animals. Although the cloned fetuses and placentas are extremely large, it is notable that animals produced as ES cell-tetraploid chimeras are intermediate in size (6). One interpretation of these data would be that a normal placenta cannot ‘‘rescue’’ the growth phenotype. It is important to remember, though, that ES cells do contribute to mesenchymal and vascular elements of the placenta (13–15). Therefore, without knowing the cellular composition of the